BIOLOGIA PLANTARUM (PRAHA) 8 (1) : 73--79, 1966

The Mechanical Transmission and Some Properties of a Disease of Cow-, Heracleum sphondylium L.

ZDENKO POL/[K Department of Plant Pathology, Institute of Experimental Botany, Czechoslovak Academy of Sciences, Praha*

Received March 1, 1965

Abstract. The described virus of cow-parsnip, Heracleum 8phondylium L., was found in three ruderal localities of Greater Praha. The symptoms are manifested by dccolorations which consist of bright yellow areas spreading from the centre of the leaf blade along the main veins. These symptoms appear severely in May. Under higher temperatures and in a chronic stage of infec- tion the symptoms are more or less masked. The disease is mechanically transmissible to , , parsnip, , sowbane, Chenopodium quinoa and C. giganteum. The author failed to transmit the disease to , , caraway and to 27 species of differential host plants, he failed in the transmission of the virus by the dodder, Cuscuta campestri8 Yu~cx., too. Thermal inactivation point of the virus lies between 51 ~ and 55 ~ C. Infectivity of extracted sap was lost after 2 days at room temperature.

In 1963, a young plant of Heracleum sphondylium L. found at Praha-Hole- ~ovice showed a type of mosaic disease caused by a virus that preliminarily appeared to differ from the biological point of view from of umbeliferous plants, described in literature. The test showed that this virus is able to infect only a limited number of plant species, including a few umbeliferous crop plants, such as parsley, coriander, parsnip and dill. Principally because of its unusual relationship to obligate differencia! host plants and the limited state of our knowledge concerning European virus diseases of umbeliferous plants, the disease was studied further. The disease is not common and has been found only rarely during routine observations of weed associations at Praha-Dejvice and Praha-Hostiva~ in 1964. Transmission trials gave evidence of identity of causative agents.

Results The host range of the virus has not been studied extensively, but most of the species tested proved to be immune from infection.

* Address: Na Kar ovce 1, Praha 6.

73 74 z. POL/kK

Species of plants found susceptible are as follows: Petroselixu~n hortense HoFr~., parsley; Petroselinura hortense var. crispum (MILL.); Coriandrum sati~uYa L., coriander; Pastinaca sativa L., parsnip; Anethum graveolens L., dill.; Chenopodium quinoa W~LD.; C. giganteum DON., and C. murale L., sowbane. Plants showing no indication of infection are listed below in alphabetical order: Amaranthus caudatus L., lovelies bleeding Apium graveolens L., celery Beta pateUaris Moq. B. vulgaris L., sugar beet Brassica naTus L., rape B. oleracea L., cabbage Capsicum anr, uum L., red pepper Ca~ urn carvi L., caraway Chenopodium polyspern, um L. Cu~urais melo L., musk melon C. sativus L., cucumber Datura innoxia MILL., downy thorn-aple D. stramonium L., jimson-weed Daucus carrota L., carrot Gomphrena globosa L., globe-amaranth Helianthu~ an~uus L., common sunflower I.upinus albus L., white lupine Medicago sativa L., alfalfa Nicandra physaloides (L.) GAERT~., apple-of-Peru Nicotiana glutinosa L. 2V. sylvestris SPEG. et COMES N. tabacum L. var. Samsun, tobacco _IV. taba(ura L. var. Xanthi-nc, tobacco Phaseolus vulgaris L., French bean Physalis floridana RYDB., ground cherry Pi~um sativum L., pea Solanum lycopersicum L., tomato S. luberosum L., potato Tetragonia expansa MURR., ~ew Zealand spinach Zinnia elegans JAcQ., zinnia It seems unlikely that any of the above plants arc symptomless carriers of the virus, as attempts to recover the virus from them have always been unsuccessful. It is necessary to stress the failure to transmit the virus to , caraway and celery. Four varieties of carrots (Amsterodamsks Chantenay, Nantais and Stupicks -- together 150 plants), two of caraway (~esk~ and Moravsk~ -- together 80 plants) and 150 celery plants were tested. Symptoms of the disease in cow-parsnip leaves The first symptoms in nature seem to be a mild chlorosis or flavescence in the terminal leaflets of the first leaf. The leaflets are deformed and often curled downwards. Later decolorations consist of bright yellow areas spread- ing from th3 centre of the leaf blade along the main veins (Fig. 1). The collapse V[P~U.~ DISEASE IN HERACLEUM SPHONDYLIUM 75 of the veins while growth is still proceeding results in a chzck to their growth, relative to that of the interveinal tissue, so that leaves present a curly or crumpled appearance with sinuate margins. Symptoms described appear severally in May. Under higher temperatures and in later stages of infection the symptoms are partly or completely masked. This phenomenon is probably due to the level of virus concentration in the plants which progressively declines. At the end of June it is impossible to distinguish between the diseased and the healthy individuals because of the loss of symptoms. At this stage the transmissibility of the disease from the source is very difficult, if not altogether impossible. There appears to be a distinct difference in the severity and symp- tom response under different substrate conditions. In humid or nitrous locali- ties there are milder changes of habitus, the disease often develops a faint mottling only in the leaflets. Symptoms in susceptible plants tested Petroselinum hortense HOFFM., parsley: The first symptoms to develop under glasshouse conditions are a clearing of veins and veinlets and blister-like elevations of the youngest leaves (symptoms in small seedlings develop 4 to 5 days after inoculation). As the disease progresses, a conspicuous vein-banding occurs. The yellow bands are at first narrow but gradually become broader and diffuse in outline. The interspaces between the veins turn yellow (Fig. 2). This occurs approximately a fortnight after inoculation. These symptoms are followed by severe deformation, leaflets are often narrow and twisted (Fig. 3). At that time, primary chlorotic lesions appear in inoculated leaves. In later stages infection becomes milder; leaves show deformation and slight decoloration only. Plants which have wintered in a glasshouse are in the following year symptomless and apparently free of virus. (Attempts to transmit the virus from wintered plants failed.) Similar symptoms of a varying degree of severity were demonstrated after the infection of four parsley varieties of Czechoslovak assortment (Dob~enick~, Hans163 KrAtks and OlomouckA E/I). The variety KrAtks losses its symptoms soon after their maximum development. Petroselinum hortense var. crisTum (MILL.): Characteristics of the disease are similar to those mentioned above. Out of 60 inoculated individuals three became infected only. Coriandrum sativum L., coriander: Systemic leaf symptoms which appear after seven days are formed by minute grey spots which coalesce forming enlarged necrotic spots. In an advanced stage of the disease severe top necrosis occurs. Plants which survive grow slowly and are severely stunted. On reaching approximately 10 em they start to flower. Transmissibility of the virus to Coriander is difficult especially to the variety coriander HrubSick~ (3 out of 30 inoculated plants were infected). Pastinaca sativa L., parsnip: Seven to nine days after infection systemic curling and puckering of the youngest leaves followed by the formation of slight diffuse yellowish green areas occur. These symptoms disappear after a fortnight. The virus was transmitted in a very low percentage (5 out of 45 plants were infected). Anethum graveolens L., dill: Systemic transverse yellowish strips in leaves appear 10 days after inoculation. Chenopodium quinoa WILLD. and C. giganteum Do~.: These species develop 76 z. POLAK local lesions on inoculated leaves 7--10 days after inoculation. The lesions appear as minute spots, later showing a light-brown dead centre (Fig. 4 and 5). They enlarge with age, but show no tendency to coalesce. Old lesions are 2--3 mm in diameter. Virus was not recovered from uninoculated leaves. C. ~urale L., sowbane: Sowbane plants form minute, necrotic local lesions. These lesions took longer to form than did the lesions on previous Cheno- Todium species. Systemic symptoms were not noted. Attempts to transmit the virus by the dodder, Cuscuta campestris YU~CXE~ Dodder was established on diseased parsleys and stems of the parasite were trained to healthy parsleys and celeries. Twenty healthy plants of each species were parazitized, but none developed symptoms of the disease. To test the ability of the dodder to acquire virus from infected host plant, juice was extracted from stems growing on an infected and parsley tested on C. quinoa leaves. Results were compared with those obtained with infectious sap from the parsley on which doder was growing. Lesions were not produced in the test with extract from the dodder. Check test was positive. Preliminary tests with an vector Green peach , Myzus persicae SULZ., fed on infectious young parsley seedlings did not transmit the virus to healthy parsley and celery plants. Further detailed studies will be carried out on virus-vector interrelations using green peach aphids and some species specific for umbeliferous plants under various lengths of acquisition and test feeding periods. Thermal inactivation point Thermal inactivation point was determined in crude infectious extract from young parsleys. T~o ml of the sap was placed in injection ampullas and subjected to the desired temperature in a water bath for 10 minutes (45 ~ 50 ~ 55 ~ 60 ~ 65 ~ and 70 ~ C). Contents of ampu]las were immediately after cooling tested on sets of 10 leaves of C. quinoa. The test indicated that the thermal inactiva- tion point lay between 51 ~ and 55 ~ C. (The experiment was twice repeated.) Longevity in vitro Longevity in vitro was studied with the same crude extract used for thermal inactivation. Infectious sap was stored in a stoppered 25 ml flask under room temperature. At 24 hours' intervals 2 ml of sap was removed and tested on 10 C. quinoa leaves. The virus retained its activity for at least two days.

Discussion From Czechoslovakia there is only a limited number of reports dealing mostly with symptoms of virus diseases ascertained in several umbeliferous crop species found in the open. None of the viruses described can be considered as related to the virus studied (BLATTN~ 1933, S~OL~K 1950, 1957). It holds good even for the mosaic disease of carrots recently studied by CHOD (1965). On the basis of our experimental results mentioned above it seems to be premature to draw any conclusions concerning the identification of the virus. Ascertained biological properties, however, enabled us to eliminate most of the furhter viruses described from or experimentally transmitted to some umbeliferous species. Because of their persistent relationship we can exclude such viruses as celery yellows -- aster yellows -- (SEvERIN 1929), celery yellow. spot (FREITAG and SEVERIN 1945C) and alfalfa witch's broom (KuNKEL 1952) VIRUS DISEASE IN HERACLEUM SPHONDYLIUM 77

and those being artificially transmitted to celery by means of a dodder, cranberry false blossom and dodder latent mosaic viruses (KSHLER and KLINKOWSKI 1954). Mechanically transmissible viruses which do not matter with regard to results of our tests on differential host plants are as follows: beet curly top virus (SEvERI• 1929, SEVE~I~ and FREITAO 1938), alfalfa mosaic virus (SNYDER and Rich 1942), tobacco ringspot virus (SEvERI~ 1950), (Bo~NEMAISO~ 1938, SEVERIN 1950) and its strains determined as south- ern celery mosaic virus (WELLMA~ 1934 a, b, c, 1935, DOOLITTLE and WELLM~ 1934) or tomato aspermy virus (SEM~L 1956 a, b), and lovage mosaic virus (SMITH and MARKHAM 1944). In host range mechanically infected, the virus differs significantly from celery calico and celery "pseudo-calico" viruses described by FRErTAG and SEVEI~nV (1939), SEVERIN (1942, 1951), SEVERIZq and FREITAG (1938), tOO. From the evidence available it is difficult to clear up the possible relationship to the remaining three viruses of umbeliferous plants, carrot mottley dwarf, poison-h~mlock ringspot and western celery mosaic (crinkle leaf strain of western celery mosaic). According to STUBBS' data (1948, 1952, 1956) the carrot mottley dwarf virus was not transmitted by aphids to parsnip, celery, caraway and parsley; on the other hand, it was successfully transmitted to dill and coriander. Symptoms of infected coriander are somewhat similar to those in our experi- ments: "ehlorosis and reddening of the foliage was accompanied by severe stunting and infected plants usually died." The host range of poison - hemlock ringspot virus (FREITAO, SEVERIN 1945b) is limited to umbeliferous hosts. Transmission of this virus by means of honeysuckle aphid was successful in celery, coriander, varieties of carrots, parsnip and varieties of parsley. Mechanically it was transmitted with dif- fienity from parsley to parsley only; other transmissions failed. Crinkle leaf strain of western celery mosaic (FgEITAG and SEVERn~ 1945a) was mechanically transmitted to dill, caraway, coriander, carrot, parsnip, parsley and celery. Th~ authors failed to transmit it to a number of non- umbeliferous species but they also failed to transmit it to cow-parsnip, Heracleum lanatum MIc~x. The thermal inactivation point of the crinkle leaf strain lies between 55 ~ and 60 ~ C (FREITAG and SEVEI~II~ succeeded to transmit the virus only in one out of seven preparations exposed for 10 minutes to 55 ~ C to one single plant out of 35 plants inoculated in every experiment). ThermM in- activation of our isolate came about between 51~ ~ C. Somewhat similar is the tolerance ascertained to againg in vitro, too; 3 days for the crinkle leaf strain and 2 days for the virus tested. Based upon the above evidence, the crinkle leaf strain of western celery mosaic seems to resemble our isolate to a certain degree more than do other two viruses. To draw any obligatory conclusions, however, it is necessary to carry out investigations on comparative symptomatology. Also virus-vector interrelations will help us to clear up this problem.

References B~TTN'2, C. : Mozaika celeru (Aplum graveolens). [Mosaic disease of celery (Apium graveolens).] -- Ochrana Rostlin 13 : 145--146, 1933. BO~NE~ISON, G.: Sur une maladie i~ virus du e61erl et du eoncombre. -- C.R. Aead. Agrie. do France 29 : 897--904, 1938. 78 Z. POL~I~

CsoD, J.: l~kter~ vlastnosti viru mozaiky mrkve. [Some properties of carrot mosaic virus.] -- Rostlinn~ V~roba, Ochrana Rostlin, in litt., 1965. DOOLITTLE, S. P., W~-~M~N, F. L.: Commelina nudi]lora a monocotyledonous host of a celery mosaic in Florida. -- Phytopathology 24:48--61, 1934. FR~ITAO, J. H., SEv~m~, H. H. P.: Additional celery viroses. -- Phytopathology 29 : 824, 1939. FREITAG, J. H., SEVERIN, H. H. F.: lnsoct transmission, host range, and properties of the crinkle-leaf strain of western-celery-mosaic virus. -- Hilgardia 16 : 361--370, 1945a. FR~.ITAG, J. H., SEW:R~, H. H. P.: Poison-hemlock-ringspot virus and its transmission by aphids to celery. -- Hilgardia 16 : 389--405, 1945b. FREIWAO, J. H., SEVE~IN, H. H. P.: Transmission of celery yelow.spot virus by the honeysuckle aphid Rhopalosiphum conii DVD. -- Hilgardia 16 : 375--384, 1945c. KOHLF.R, E., KLINKOWS~I, M.: Viruskrankheiten. -- Handbuch der Pflanzenkrankheiten, Berlin und Hamburg, 1954. KUN~EL, L. O.: Transmission of alfalfa witch's broom to non-leguminous plants by dodder, and cure in Periwinkle by heat. -- Phytopathology 42 : 27--31, 1952. SE~L, J.: A virus of celery related to Cucumis virus 1st. Chr. Noordam. -- T. Plantenziekten. 62 : 177--178, 1956a. SE~A~., J.: Note sur la pr6senco choz le e616ri (Apium graveolens) de Cucumis virus 1st. Chr. Noordam. -- Parasitica 12 : 29--31, 1956b. SEVEnI~, H. H. P.: YeUows disease of celery, lettuce, and other plants, transmitted by Cicadula sexnotata (F~L~.). -- Hilgardia 3 : 543--583, 1929. SEVE~IN, H. H. P.: Celery calico on perennial delphiniums and certain other host-plants. -- Hilgardia 14 : 441--445, 1942. S~VERIN, H. H. P.: Symptoms of cucumber mosaic and tobacco ring spot virus on celery. -- Hilgardia 20 : 267--277, 1950. SEVV.~IN, H. H. P.: Symptoms of celery-calico virus on tomato plants. -- Hilgardia 20 : 137-- 145, 1951. S~'v~.RI~, H. H. P., FRErrxo, J. H.: Western celery mosaic. -- Hi]gardia 11 : 493--558, 1938. SMITH, K. M., MarKHAm, R.: A virus disease of lovage. -- Phytopathology 84 : 335--340, 1944. S~OL~,K, J.: Mosaiky pastin~ku. [Mosaic diseases of parsnip.] -- Ochrana Rostlin 23 : 272, 1950. S~OL~,K, J.: Virov~ deformita celeru. [Virus deformity of celery.] -- Sbor. vys. ~ik. zem. v Praze: 181-- 188, 1957. SNYDER, W. C., RICH, S. : Mosaic of celery caused by the virus of alfalfa mosaic. ~ Phy%o. pathology 32 : 537--539, 1942. STUBBS, L. L.: A new virus disease of carrots: its transmission, host range, and control. -- Austr. J. Sci. Res. B ! : 303--332, 1948. STUBBS, L. L.: Further host range and transmission studies with a virus disease of carrots endemic in Australia. -- Austr. J. Sci. Res. B. 5 : 399--408, 1952. STUBBS, L. L.: Mottley dwarf virus disease of carrot in California. -- Plant ]:)is. Reporter 40 : 763--764, 1956. WEL~MAN, F. L.: A disease of banana markedly similar to bunchy top, produced by celery virus 1 in U.S.A. -- Phytopathology 24 : 1032--34, 1934a. WE~L~, F. L.: Identification of celery virus 1, the cause of southern celery mosaic. -- Phyto- pathology 24 : 695--725, 1934b. WELLMAN, F. L.: Infection of Zea mays and various other Gramineae by the celery virus in Florida. -- Phytopathology 24 : 1035--37, 1934c. Wv.~A~, F. L. : Dissemination of southern-celery mosaic virus on vegetable crops in Florida. Phytopathology 25 : 289--308, 1935.

Z. Pop,x, odd~lenl fytopatologie ~stavu experiment~lnl boteniky ~SAV, Praha: Meeha- nlek~ p~enosy a n~kter~ vlaslnosti virov~ho oehurav~ni bol~evniku, Heracleum sphondylium L. Biol. Plant. 7 : 73--79, 1966. Ochurav~nl se projevuje na listech ~lutozelen~Tni nebo ~lut~mi skvrnami, ~ii~iclmi so pedal nervatury vy~w ~du. Tyto p~iznaky jsou zvl~t~ dob~e patrn6 v kv~tnu. P~i vy~ich teplo- t~ch a v ehronick6 f~zi ochurav~ni p~iznaky mizl. Zjistili jsme, ~e ochurav~ni jo mechanicky p~enosn6 na petr~.el, koriandr, pastins a kopr. U petr~elo vyvol&v~ ncjprve prosv~tlent nervatury, posl~ze chlor6zu st~odni 5s listkfi, pro- v~zenou t~kou deformaci. Koriandr reagujo na infokci odumir~nim nejmladw listfi a posl~ze cel~ho vegetaSniho vrcholu; rostliny, jim~ vrcholy neuhynuly, vyrfistaji v zakrsl6 jedinco asi jen 10 cm vysok~. U pastins se projevuje infokee lehkou zelenou skvrnitostl, nejSast~ji v~k zkrabat~nlm nejmladw listfi, kter~ pozd~ji pHznaky ztr~cejl. Na llstclch kopru so vytvo~i VIRUS DISEASE IN HERACLEUM SPHONDYLIUM 79 drobn6 tlut~ p~iSn~ prou~.ky a 6s P}enos viru so poda~il jew na 3 druhy rodu Cheno~odiurn, Ch. quinoa, Ch. giganteum ~ Ch. murale, kter~ reaguji tvorbou lok~lnich nokrotick~rch lesL Virus so nopoda~ilo p}on~st na color, turkey, kmin a 27 dii~oren6nich hostitolsk~'ch druh5. Pokusy o p~onos viru kokotici, Cuscuta carnpestris, nebyly fisp~w Bylo dale zjiw to torm~lnt inaktiva6ni bod viru lo~.i mezi 51--55~ C a ~o virus ztrttci svoji aktivitu v surov~ ~s p~i laboratornl teplot~ ji~. po 2 dnech.

3. IIo~, OT~le~enHe ~pnTOnaTo~or~]~ I/IHCTI~TyTa ~gcnep~MeHTa~bHO~ 60TaHI~]~ qCAH, Hpara: Mexmmqecga~ nepepaqa H neKoTopbie CBO~CTBa nnpycnoro 3aSo~enauHn Heracteum sphondylium L. -- Biol. Plant. 7 : 73--79, 1966. 3a6o~eBan~e Hpo~BJI~eTC~ no~HaeHHeM meJiwo-3eJ]eHMx B~H me~TMX nffTeH pacnpo- cTpaHamm~xca no xo;Iy HepBaTyp~ B~cmero nopn~Ka. IIpnanaKH ~TH xopomo 3aMeTHhI n Mae. l-Ipli 6oaee H~CO~HX TeMnepaTypax ~ B XpOHH~ecKofi CTa~IH 3a6oae~anH~ OH~ Ilcqe 3aIOT. B~ao ycTaHo~eno, qTO aaSo~eHan~e Mexan~,~ec~M nyTeM nepe~laeTc~ neTpym~e, ~op~amlpy, nacT~Hai~y ~ y~pony. u neTpym~H ona n~3~HaeT nocHeTaeHHe nepHaTyp~I xaopo3 cpe~nefi '4acwI4 7iI4CTa, conpoBo~aeM~e T~eaofi ~edpopMa~I~cfi JlI~cTbeH. Hop~amlp pear~pyeT Ha nn~e~nm OWMnpaHI~eM r[ ne~poaoM Ha~6oaee MOaO~I~X a~CT],eH a 3aTeM H BepxymeqHofi TO~ pocTa. Y nacw~Ha~a ~n/~e~ oSHapym~Haewc~ aer~ofi 8e~eHofi HgTHI4CTOCTbIO, HO ~ame Bcero MopmI4HI4qTOCTI~IO nanToaee MO~O~x JIl~lt'WbeB. ~br y~pona ofpaaoe~HaIOTCg Meanne meJ~w~e nonepeqn~e noaoc~n n qepwoq~m I/1H~C~OI~m eme y~aaocs B~aHaT], na Tpex Hmlax Chenopodium -- Ch. quinoa, Ch. giganteum n Ch. murale- 06pa- 3OBIaIBaJlIICb TOJII~KO MeCTHI~e nonpem~eng~ no cncTcMnoe aaTo~e~anne BI~anano He 6hl~IO. B~pyc He y~Iano('b nepe~law], ceabuepcm, uop~rn n TMBHy ~ 27 Bn~laM ~li~p~pepenltl~aabHUX pacTeH~U -- xoaael. Bnpyc He y~la~o('b nepene('Tn npn nOMOmH Cuscuta campestris. B~,qo y('waHoszeno ,~WO xepMa~]bnan nna~Tnra~tnn ~npy('a npom'xo~lnT ~tcm~ly 5t ~ 55 ~ C. IIpn ~r weMnepaType nnpyc nna~TnBnpyeTc~ qepea asa ~In~.