Overwintering and monitoring of potato leafroll virus in some wild crucifers

1993) 505

O V E R W I N T E R I N G AND M O N I T O R I N G OF POTATO L E A F R O L L V I R U S IN S O M E W I L D C R U C I F E R S 1

Lee Fox 2, K. Duane Biever 2, H. Harold Toba 2, James E. Duffus 3, and Peter E. Thomas 4

Abstract A potato leafroll virus (PLRV) isolate has been successfully transmitted

to and recovered from two wild crucifers, Sisymbrium altissimum L. (J im Hill or tumble mustard) and Capsdla bursa-pastoris (L.) Medic. (shepherd's purse) by the green peach aphid (GPA), Myzus persicae (Sulzer). Virus antigen in both plant species was found to be higher in root tissue than in foliar tissue, based on enzyme-linked immunosorbent assay (ELISA) determinations. C. bursa-pastoris was apparently a relatively poorer source of inoculum for the GPA than S. altissimum. Using two geographically-separated biotypes of C. bursa-pas~ris, a Washington biotype was found to contain higher antigen titer in both leaf and root tissue than a California biotype, as determined by ELISA. Field studies demonstrated that both weed species can serve as overwintering sources of PLRV.

C o m p e n d i o

Un aislamiento del virus del enrollamiento de la hoja de la papa (PLRV) ha sido exitosamente transmitido y recobrado de dos crucfferas silvestres, Sisymbrium altissimum L. (J im Hill o mostaza postrada) y Capsella bursa-paag~ (L.) Medic. (bolsa de pastor) por el ~/ido verde del melocotonero (GPA), Myzus persicae (Sulzer). Bas~ndose en determinaciones efectuadas con la prueba inmunol6gica ELISA se encontr6 que el antfgeno del virus en ambas especies vegetales era m~s alto en los tejidos de las ra~ces que en los de las hojas. C. bursa-pastoris fue aparentemente una fuente m~ts o menos pobre de in6culo para el GPA, en comparaci6n con S. a/t/~rnum. Utilizando dos biotipos geogrgtficamente separados de C. bursa-pastoris, se encontr6 que

1Mention of a trademark or proprietary product does not constitute a guarantee or war- ranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. This article reports the results of research only. 2Biological Research Technician and Research Entomologists, respectively, USDA, ARS, Yakima Agricultural Research Laboratory, Yakima, WA 98902. 3Research Plant Pathologist, USDA, ARS, U.S. Agricultural Research Station, Salinas, CA 93905. 4Research Plant Pathologist, USDA, ARS, Irrigated Agriculture Research and Exten- sion Center, Prosser, WA 99350. Accepted for publication December 14, 1992. ADDITIONAL KEY WORDS: Myzuspersicae, green peach aphid, ELISA, enzyme-linked immunosorbent assay, Sisymbn'um altissimum, Capsella bursa-pastoris, virus transmission, potato virus, aphid vector, weed host.

506 AMERICAN POTATO JOURNAL (Vol. 70

un biotipo de Washington tenfa, tanto en los tejidos de la hoja como en los de la rMz, una concentraci6n m~is aha de antfgeno que un biotipo de California, tal como qued6 comprobado con la t6cnica ELISA. Estudios de campo demostraron que ambas malezas pueden servir como fuentes de invernaci6n del PLRV.

Introduction

Literature on potato leafro11 virus (PLRV) is extensive but lacks experimental data to characterize the seasonal epidemiology of this patho- gen. PLRV has been considered the causative agent of one of the oldest known virus diseases of plants, and its association with phloem necrosis in potato tubers is established (12). Non-solanaceous hosts of PLRV include members of Amaranthaceae, Cruciferae, Nolanaceae and Protulacaceae, and only shepherd's purse, Capsella bursa-pastoris (L.) Medic., in the Cruciferae (7).

Thomas (15) reported that in central Washington State, volunteer potato plants from outside the commercial potato fields are the chief source of both leafroll primary inoculum and the aphid vector and that annual weeds are probably not a major source of primary inoculum. In Maine, weeds were reported to be an important source of aphids entering commercial potato fields, but they were not believed to host PLRV (13).

The objective of the field phase of this study was to determine if two cruciferous weed species that are common and prolific in the potato growing areas of the Pacific Northwest could harbor PLRV through the winter and serve as an inoculum source for green peach aphid (GPA), Myzusper- s#ae (Sulzer), the following spring. In the laboratory phase, particular emphasis was placed on differentiating between results obtained from enzyme-linked immunosorbent assay (ELISA) determinations of host plants and aphid transmission bioassays from host plants to indicator plants as they relate to specific host plant species. Also, tests were initiated to determine if C. bursa-pas~nk selections from different geographical origins express similar reactions to PLRV infection and what effect this has on ELISA detection results and aphid vectoring capability.

Mater ia ls and Methods

Characterization of PLRV Isolate 2243

The potato leafroll virus isolate (PLRV-2243) used in this study was taken from Solarium tuberosum L. cv. Russet Burbank; it causes primary foliar leafroll symptoms and also net necrosis in some progeny tubers of this cul- tivar. Negative ELISA reactions result when infected plant tissue samples are tested against beet western yellows virus (BWYV) antiserum.

PLRV-2243 was propagated in Datura tatda L. grown in greenhouse soil beds. Plants were inoculated as seedlings using GPA. When infected

1993) FOX, et al: POTATO LEAFROLL VIRUS 507

plants reached a height of 45-90 cm, leaves were harvested and virus puri- fied according to the method of Hassan and Thomas (8). This purification procedure yields antisera that does not react to virus-stress antigen which can result in false positive PLRV ELISA readings (6). A polyclonal antiserum to PLRV-2243 was produced in rabbit and used in the double antibody sandwich method of microplate ELISA (2).

ELISA Procedure

For ELISA, )'-globulin obtained from PLRV-2243 rabbit antiserum was conjugated to alkaline phosphatase (2). Coating globulin diluted 1:2500 was incubated for 2 hr at 37 C. Plant tissue samples were triturated with mortar and pestle in 1:20 (w/w) extraction buffer (phosphate-buffered saline containing 2.0% polyvinylpyrrolidone [MW 40,000] [w/v], 0.2 % ovalbu- min [w/v], and 0.05% Tween-20 [w/v]). Homogenates were stored in glass culture tubes at room temperature for up to 4 hr. Then 200-gl aliquots were added to each well in coated polystyrene Micro ELISA Immulon II | plates, (Dynatech Laboratories, Alexandria, VA) and incubated overnight at room temperature. Conjugated )'-globulin diluted 1:2500 was incubated for 2.5 hr at room temperature. Substrate (p-Nitrophenyl phosphate, Sigma 104 | was added to each ELISA plate well (250/A) at a concentration of 1 mg/ml in diethanolamine, HC1 buffer, pH 9.8 and the plates were incubated at room temperature for 30 min. Absorbance values (A405) were determined by scanning the plates in a Titertek Multiskan MC | spectrophotometer (Flow Laboratories, McLean, VA). Extraction buffer alone and with PLRV- free plant tissue (same type as being tested) were used as negative controls. Leaf tissue from greenhouse grown Physalisfloridana Rydb. infected with PLRV-2243 was used as the positive standard. The absorbance values for three to five samples were averaged.

Nctoring Efficiency of M. persicaefrom the Stock Colony

All aphids used in transmission bioassays and field plant inoculations were taken from GPA colonies maintained in the insectary on either virus- free or PLRV-2243 infected P f/or~na. To determine their vectoring efficiency, a single aphid (third or fourth instar) from the infected colony was placed on a virus-free P. flork/ana seedling (two true-leaf stage) and held in a growth chamber for 48 hr. Plants with dead or missing aphids following feeding period were eliminated from the test and 100 of the remaining plants were fumigated with methyl bromide (22.77 g/m 3 for 2 hr at ca 21 C) to kill the aphids. Plants were then placed in an insect-free greenhouse for 2-3 wk before virus symptoms were recorded and leaf samples collected for ELISA determinations.

Plant Host Range of PLRV-2243

PLRV-2243 plant host range studies were carried out by introducing five to eight viruliferous GPA (third or fourth instar) to each test plant seed-


ling. The aphids were left on the plant for 48 hr, then survival was determined; plants with less than four surviving aphids were eliminated. The remaining plants were fumigated with methyl bromide (22.77 g/m 3 for 2 hr at ca 21 C) to kill the aphids. At various intervals after the initial inoculation, attempts were made to recover virus from the test plants. Three excised leaves (from top, middle, and lower regions of each test plant) were placed in a petri dish and three virus-free GPA were placed on each leaf (9 aphids/petri dish). After a 48-72 hr acquisition period, two aphids from each excised leaf were transferred to a P. florMana seedling (6 aphids/plant) for 48 hr. Plants were then fumigated with methyl bromide to kill the aphids. Virus symptoms expressed by the indicator plant were recorded over 2-3 wk. ELISA was used to detect virus antigen in the leaf and root tissues of the test and indicator plants.

Laboratory 7?ansmission of PLRV to Wild Crucifers

Based on the preliminary findings of a PLRV host range screening, two winter annual weed species were selected for further evaluation. Young (two true-leaf stage) Sisymbrium altissimum L. (Jim Hill or tumble mustard) and C. bursa-pastoris (including two geographically-separated biotypes) were inoculated with PLRV by introducing five to eight viruliferous aphids (third or fourth instar) to each plant. Plants with less than four live aphids after 48 hr were discarded. The remaining plants were fumigated with methyl bromide to kill the aphids then held in a greenhouse until assayed. S. a/t/s- simum leaf samples for ELISA determinations were collected 23, 40, 88, and 94 days after inoculation. At day 94, two additional samples were taken: root tissue for ELISA and leaves for verification tests. The C. bursa-pastoris biotypes were grown from seeds collected at Salinas, California (CA biotype) and Yakima, Washington (WA biotype). For both biotypes, leaf samples were taken for ELISA at post-inoculation day 20 and root samples at day 41, and leaves for verification tests at day 41. ELISA samples from each S. altissimum plant were replicated in five wells of the polystyrene plates and from each C. bursa-pastoris plant in three wells. In verification tests, GPA transmission bioassays were made to P. flork/ana indicator plants, which were evaluated for infection by visual symptoms and ELISA at 6 wk after inoculation. ELISA samples from each indicator plant were replicated in three plate wells.

Overwintering of PLRV in Wild Crucifers

Two field sites in central Washington were monitored to determine if PLRV can survive in naturally occurring overwintering populations of C. bursa-pastoris and S. altissimum. At a location near George, both weed species survived the winter (1988-89) in segregated plant communities near the outer periphery of a 1988 commercial potato planting. The potato field was under a circular center-pivot irrigation system with C. bursa-pastoris present only


in the area immediately outside the potato planting, receiving some water, but less than the potatoes. S. altissimum was present in the noncultivated area of the arid shrub-steppe surroundings, beginning 40-50 m from the potato field and beyond water from the sprinkler. The potato planting had been naturally infected with PLRV early in the season as shown by current season (primary) leafroll foliar symptoms in many plants at harvest. The following year, on 17 March, when the plants were entering the reproductive growth stage, root samples from 50 C. bursa-pastoris and 100 S. altissimum surviving plants were collected for ELISA to determine if they were infected with PLRV. ELISA samples from each plant were replicated in three plate wells.

The overwintering study site at the USDA ARS Yakima Agricultural Research Laboratory Field Station, located 17 km east of Yakima, was monitored over two consecutive winters. In the first year, 50 S. altissimum plants were selected from the natural population present in a non-cultivated 5 X 180 m area bordering a potato planting. On 25 OCtober 1988, the plants were in a prostrate vegetative stage of growth (5-6 wk post-emergence) when 20-30 apterous viruliferous GPA were introduced per plant. Each plant had at least five aphids present on the day following aphid introduction. The expected low temperatures over the next few weeks were relied on to kill the aphids. Plants were not caged because of concerns about further alter- ing plant growth and ultimately affecting the overwintering survivial of the plants. On 9 May 1989 (195 days following initial inoculation), surviving plants were examined for aphids and leaf samples were collected for a transmission bioassay. The plants were 40-70 cm tall and in their reproductive stage of growth.

In the second year, 50 S. altissirnum plants were inoculated and 50 were left uninoculated. Leaf samples for a transmission bioassay were collected from each of the 100 plants 4 hr before 50 receiving viruliferous aphids. Plants were inoculated on 11 October 1989, 2 wk earlier than the previous year but with no noticeable difference in plant size. Selected plants were marked for indentification the following spring and were separated from each other by at least 3 m. On 21 March 1990 (161 days following initial inoculation), leaf samples were taken for a transmission bioassay from overwintering plants. In both years, transmission bioassays were made to P. f/orh4ana indicator plants using GPA. At 4 wk after inoculation, the indicator plants were evaluated for infection by visual symptoms and ELISA. ELISA samples from each plant were replicated in three plate wells.

Statistical Analysis

ELISA absorbance values of plant tissues were analyzed by t-test and analysis of variance (10). The means were separated by Duncan's (5) multiple range test.


Results

l,~ctoring Efficiency of M. persicae from the Stock Colony PLRV was transmitted successfully by 96 of the 100 GPA (third or

fourth instar) taken from infected P. f/0ru/a~, where they had been reared, and transferred to virus-free P. flor~dana seedlings (1/plant). Infected indicator plants produced acute symptoms and leaf samples gave positive ELISA reactions. The four negative plants showed neither symptoms nor positive ELISA values. The results indicate a very high vectoring efficiency of PLRV- 2243 by aphids from the stock colony, the source of apterous viruliferous aphids and inoculum for the other laboratory and field tests reported here.

Plant Host Range of PLRV2243

Aphid transmission bioassays and ELISA results indicated the following species of plants to be susceptible to infection by PLRV-2243: CRUCIFERAE: Capsella bursa-pastoris (L.) Medic.; Sisymbrium altissirnum L.; SOLANACEAE: Datura stramonium L.; Datura tatula (L.) Torr.; Lycopersicon esculentum (Mill.); Physalis floridana Rydb.; Sola~m sarrachoides Sendt.; Solanum tuberosum L.

Species that showed no symptoms, and from which no virus was detected by either ELISA or aphid transmission were as follows: AMARAN- THACEAE: Amaranthus retroflexus L.; CRUCIFERAE: Brassica napus cv. Topas; Brassica napus cv. Dwark Essex; Raphanus sativus L., Sisymbrium loeselii L.; CHENOPODIACEAE: Beta vulgaris L.; CONVOLVULACEAE: Con- volvulus arvensis L.; SOLANACEAE: Solanum dulcamara L.

Laboratory 7?ansmission of PLRV to Wild Crucifers

Virus-inoculated S. altissimum remained in the rosette stage throughout the test period and exhibited no visual virus symptoms. The mean absorbance values from ELISA indicate antigen was not evenly distributed between root and leaf tissue (Table 1) with roots having the higher titer, as reported by Casper (1) for potato and P. f/or~na. ELISA results obtained with foliage samples gave some indications of infection, but they were not reliable because of inconsistencies. Out of 20 inoculated plants, the number of plants having absorbance values from leaf tissue samples that were significantly greater than mean values for control plants were 5, 5, 13 and 3 plants at 23, 40, 88 and 94 days following inoculation, respectively. On the other hand, root tissue samples from 19 plants had absorbance values (0.439 - 1.402) significantly greater than the mean value of control plants (0.103). Furthermore, positive visual foliage symptoms and ELISA readings from leaf tissue sample of P. f/ork/ana indicator plants were obtained following aphid transmissions from these same plants. Positive virus transmission to indicator plants was indicated by ELISA values of 0.199 to 0.491 compared to 0.104 in the single plant with negative transmission.


TABLE 1.--Enzyme-linked immunosorbent assay (ELISA) absorbance values (4,05) following laboratory transmission of P L R V by Myzus persicae to two crucifer weeds.

S. altissimum C. bursa-pastoris Plant biotype Plant biotype CA WA CA WA

Leaf Root Leaf Root Number of days following inoculation

Treatment 23 40 88 94 94 20 20 41 41

Inoculated Mean a 0 .137 0.123 0.176 0.144 0.941 0.213 0.485 b 0.381 0.814 b +S.E. 0 .002 0.005 0.008 0.003 0.031 0.013 0.018 0.021 0.031

Control Mean a 0 .111 0.096 0.105 0.106 0.103 0.111 0.121 0.100 0.114 +S.E. 0 .002 0.005 0.009 0.004 0.003 0 0 0 .003 0.005

r

an ffi 20 inoculated plants and 5 control plants. Samples from each plant were replicated in five ELISA plate wells for S. a/t/~s/mum and three wells for C. bursa-pastoru. bMean absorbance values from leaf and root samples of WA biotype were significantly greater than those of CA biotype (P ffi <0.05; student's t-test).

No virus symptoms were expressed in any C. bursa-pastoris plants on which viruliferous aphids had been introduced. However, the two biotypes grown under the same conditions exhibited somewhat different growth habits. The WA biotype remained in the rosette stage of growth longer (3- 4 wk) than the CA biotype, thereby delaying seed production. Another difference was significantly higher (t-test, P < 0.01) absorbance values for leaf and root tissue samples from plants of the WA biotype than those of the CA biotype (Table 1). Here again, as with S. a / t ~ , absorbance values were higher for root samples than for leaf tissue samples. Values for root samples indicated infection in 17 out of 20 CA biotype plants, and all 20 of the WA biotype plants. However, in verification tests, GPA were able to transmit the virus to P f/0rk/ana indicator plants in only 25% (5/20) and 45 % (9/20) of the cases, respectively, based on visual symptoms and absorbance values for leaf tissues.

Overwintering of P L R V in Wild Crucifers

ELISA results from the field-collected root samples (George field site) indicated that 8 of 50 C. bursa-pastoris field plants that had overwintered were infected (Table 2). M e a n absorbance values from infected root samples ranged from 0.235 to 0.347 with the remaining 42 samples ranging from 0.069 to 0.100. Since the plants were collected 17 March 1989 before vectoring aphids were present, they had to be have received the initial inoculation the previous fall. Similar root samples from 100 S. altissimum plants


TABLE 2.--ELISA detection of P L R V in root samples from Capsella bursa- pastoris and Sisymbrium altissimum that had overwintered (1988-89) in an open field near George, Washington.

No. PLRV Total number infected Percent of plants

Plant species of plants tested plants a infected with P L R V

C. bursa pastoris 50 8 16.0 S. altissimum 100 0 0

aBased on mean absorbance values; samples from each plant were replicated in three plate wells.

resulted in negative ELISA values ranging from 0.062 to 0.081 indicating none of the plants were infected with PLRV.

At the Yakima USDA Field Station overwintering site, the 1988-89 test showed that 34% (17/50) of the S. altissimum field plants inoculated with PLRV in the fall of 1988 survived the winter (Table 3). Six of the surviving plants were hosting PLRV, based on transmission bioassay to Pflor~na. Absorbance values of leaf samples from the indicator plants, which also exhibited positive foliar symptoms, ranged from 0.462 to 0.620, while the remaining plants ranged from 0.063 to 0.073. In the 1989-90 test, none of the 100 selected field plants were naturally infected with PLRV prior to the time 50 received viruliferous aphids (11 October 1989). Of the inoculated plants, 36% (18/50) survived the winter. Only five of the surviving plants had retained the virus over 161 days following the introduction of viruliferous GPA. P f/orkk~na plants indicating infection exhibited positive foliar symptoms and absorbance values of 0.746-0.969, compared to values of 0.045-0.053 from non-infected plants. Of the uninoculated plants, 62.0% (31/50) survived the winter, of which two were found to be infected. The indicator plants exhibited positive foliar symptoms and absorbance values of 0.748-0.758 from infected plants, compared to 0.042-0.049 from non-infected plants.

Discussion

In the Yakima Valley and Columbia Basin of central Washington State, GPA primarily exhibits a heteroecious holocyclic type life cycle, overwintering in the egg stage on peach trees, Prunus persiea (L.) Batsch. The eggs hatch in mid-February to early March and winged migrants begin leav- ing the trees in late April or early May (3, 14). Peach is not a host of PLRV (9), so for GPA to disseminate the virus, it must be acquired from infected potato or alternate host plants. Both crucifers in this study, which have been shown to be overwintering hosts of the virus, commonly grow as annuals and winter annuals in this area. They are also excellent GPA host plants

1993) FOX, et ak POTATO LEAFROLL VIRUS 513

TABLE 3 . - Ooerwinlering survival of PLRV and Sisymbrium altissimum in an open field area at the USDA Yakima Agricultural Research Laboratory Field Station, 27 km east of Yakima, Washington.

Winter No. plants No. plants % Survivors Treatment of selected survived infected a

PLRV inoculated 1988-89 50 17 35.3 PLRV inoculated 1989-90 50 18 27.8 Not inoculated 1989-90 50 31 6.4

aDetermined from positive foliage symptoms and ELISA results of leaf samples from P. J/0r/dana indicator plants following aphid transmission bioassays.

in nature and are among the first plants available for winged forms emigrat- ing from peach orchards. These plants can therefore serve as virus sources for aphids in the spring and intermediate hosts of migrant aphids and the virus throughout the year for the dissemination of the virus to other susceptible plants, including potato.

We have demonstrated that GPA vectoring efficiency of PLRV is determined, in part, by the host plant species and biotypes. Laboratory results indicate that GPA vectoring efficiency was greater when S. a/t/~dmum served as the PLRV source plant compared to C. bursa-pastoris. The reason for this is uncertain because ELISA absorbance values for leaf tissue samples from PLRV-infected S. altissimum tended to be lower (indicating lower virus titer) than similar C. bursa-pastoris samples, although the virus titer in root samples from plants of both species was high. Likewise, in testing the suscepti- bility of two biotypes of C. bursa-pastoris to PLRV, prompted by differences in severity of symptom expression caused by beet western yellows virus infection in two C. bursa-pastoris biotypes (4), we found that the infection rates differed when aphid transmissions were made to P . ~ . One presump- tion for these differences is the inability of the vector to acquire or transmit the virus.

ELISA can be a very accurate and definitive tool once the best tissue sampling (e.g., roots versus leaf) protocols are worked out for the plant species of interest. For example, once the root area ofS. altissimum was iden- tified as having the highest and most consistent PLRV titer, ELISA was as accurate as the aphid transmission bioassay to indicator plants in verification tests for detecting the virus. However, with C. b u r s a - p ~ , the number of plants with significantly high virus titer in root samples did not agree with the number of positive indicator plants in verification tests. Aphid transmission bioassays are apparently affected by certain host plant combina- tions, but are still needed to confirm ELISA results of most virus-plant interactions. In epidemiological studies, the ability of vectors to transmit the virus is of primary importance.


Peters and Jones (11) repor ted that no evidence exists to indicate that hosts other than potato act as a P L R V reservoir in the tempera te regions. We have demonstrated here that P L R V can and does overwinter in C. bursa- pastor/~ and S. altissimum unde r Washington conditions. At the U S D A Field Station, where S. altissimum was tested, February was the coldest month dur- ing the 1988-89 winter with an average m i n i m u m tempera tu re of -8.3 C and a -20 C on the coldest day. T h e following year (1989-90), December was the coldest mon th with an average m i n i m u m tempera tu re of -4 .0 C, but the coldest day was in February with -12.8 C. Th e overwintering results repor ted here may have impor tan t implications for the epidemiology of P L R V in the potato growing areas of central Washington. Therefore, studies should be expanded to de te rmine the occurrence of P L R V in other weed hosts, especially those that may serve as overwinter ing virus sources. Th e somewhat l imited results f rom virus acquisit ion and inoculat ion studies in the labora tory may only remotely approximate what is happening in a natural setting and obviously cannot be accepted as represent ing the vectoring ability of a GPA popula t ion on P L R V epidemiology. T h e fact that such variat ion in vector ing ability was demons t ra ted experimental ly warrants fur ther investigation unde r natural conditions.

A c k n o w l e d g m e n t s

T h e authors wish to thank Rober t A11mendinger and J e a n n e Nelson for technical assistance in both the labora tory and field phase of this proj- ect, and M a r k Weiss for assistance in statistical analyses.

Literature Cited

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3. Davis, E.W. and B.J. Landis. 1951. Life history of the green peach aphid on peach and its relation to the aphid problem on potatoes in Washington. J Econ Entomol 44:586-590.

4. Duffus, J.E. 1964. Host relationships of beet western yellows virus strains. Phytopathol 54:736-748.

5. Duncan, D.B. 1955. Multiple range and multiple F tests. Biometrics 11:1-42. 6. Gunn, L.V. and R.D. Pares. 1988. Effect of potato physiology on the interpretation

of ELISA results for potato leafroll virus. Plant Pathology 37:516-521. 7. Harrison, B.D. 1984. Potato leafroll virus. CMI/AAB Descriptions of Plant Viruses

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10. Hintze, J.L. 1987. Number Cruncher Statistical System, Version 5.01 Kaysville, Utah. 11. Peters, 19. and R.A.C. Jones. 1981. Potato leafroll virus, p. 68-70 In: Compendium

of Potato Diseases (W.J. Hooker, Ed.). Am Phytopath Soc, St. Paul, MN., 125 pp. 12. Quanjer, H.M., H.A.A. Van der Lek andJ.O. Botjes. 1916. Aard, Verspreidingswijze

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13. Simpson, G.W., W.A. Shands and O.L. Wyman. 1945. Weeds and the aphid-leafroll problem in potatoes. Maine Ext Bull No 333, 20 p.

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Documents

Overwintering and monitoring of potato leafroll virus in some wild crucifers