Seed Transmission of Plant Viruses

SEED TRANSMISSION OF PLANT VIRUSES

C. W.

Bennett

Agricultural Research Station, Craps Research Division, U. S. Department of Agriculture, Salinas, California

I. Introduction. ........................................................... 221 11. Seedling Infection by Virus Carried Outside the Embryo. ................ 222 222 A. Virus Carried on the Surface of Seeds. ............................... B. Virus Carried in Parts of Seed outside the Embryo. .................. 223 111. Transmission of Viruses Carried in the Embryo.. ........................ 226 A. Methods of Embryo Infection. .................. .................. 226 B. Types of Viruses That Are Seed Transmitted. ... .................. 236 C. Influence of Environmental Factors on Seed Transmission. . . . . . 236 D. Influence of Host Plant and Virus on Seed Transmission.. ............ 238 E. Longevity of Viruses in Seeds. ....................................... 242 F. Effect of Seed Transmitted Virus on Seedlings.. ...................... 245 G. Economic Importance of Seed Transmission of Viruses.. . . . . . . . . . . . . . . 247 IV. Factors Influencing or Determining Seed Transmission.. ................. 250 A. Inactivation of Virus in the Embryo.. ................................ 250 B. Sterility of Gametes ..................................... 251 C. Susceptibility of Gametes to Infection.. .............................. 252 D. Protection of the Embryo from Virus Infection. ................. 256 E. Conclusions. ......................................................... 258 References.. ............................................................ 258

I. INTRODUCTION One of the most characteristic and interesting relationships of plant viruses to their host plants is the high degree of protection possessed by embryos of seeds against invasion by viruses that affect the mother plant. Despite this protection, however, an appreciable number of viruses have been found to pass from one generation to the next through the medium of the seed. Until recently, it was generally assumed that where seed transmission occurred it was usually limited to a relatively small percentage of the seeds of affected plants. This still seems t o be true for most types of seed-transmitted viruses, but more recent reports, especially those by Lister (1960), Lister and Murant (1967), and Murant and Lister (1967), indicate frequent occurrence of high percentages of seed transmission of nematode-transmitted viruses in a number of susceptible plants. Fortunately, in the great majority of instances, the seed offers a highly effective barrier to passage of most viruses from one generation to the next and perpetuation of most of the more destructive viruses is still dependent on repeated infection of successive generation of plants through the agency of natural vectors. 221

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C. W. BENNETT

Nevertheless, in the case of several viruses that are seed transmitted, virus transfer through seeds is highly significant in virus preservation and spread and, consequently, in the epidemiology of the diseases they induce. In this article an attempt has been made to review the literature dealing with a rather wide range of aspects relating to seed transmission as they have been elucidated over a period of more than 50 years, but with special reference to the more recent developments in this field.

11. SEEDLING INFECTION BY VIRUSCARRIED OUTSIDE

THE

EMBRYO

A . Virus Carried on the Surface of Seeds

It is obvious that seeds from systemically infected plants might well carry virus as a surface contaminant. This would be especially true of seeds extracted from fleshy or pulpy fruits such as tomato, cantaloupe, watermelon, or cucumber. However, to be transmitted to the next generation, it would be necessary for the virus to remain active on the surface of the seeds until they germinate and then to find a method of entering the seedling in an early stage of its development. One of the very few viruses that could qualify for transmission of this type is that causing tobacco mosaic. There is evidence, as would be expected from its known properties, that tobacco mosaic virus can remain active on the surface of seeds for periods ranging from a few days to a few years. John and Sova (1955) state that tomato seeds harvested from diseased plants in 1950, 1951, and 1952, and held in bags in common storage, retained virus in 1953 with no evidence of great loss of virus in storage. Although it is assumed that virus was carried on the seed in this test, the possibility that it was carried in the seed does not appear to have been eliminated. Raychaudhuri (1952) found shorter periods of survival of tobacco mosaic virus on tomato seeds. A strain of virus associated with internal browning of tomato was active for only 1 to 2 weeks and another strain of the same virus retained activity for less than 5 weeks. However, Alexander (1960) found that, although the amount of tobacco mosaic virus associated with tomato seed declined rapidly after 1 year of storage, it could be recovered after 3 years. It seems evident from these results that tobacco mosaic virus can retain activity in association with tomato seeds for relatively long periods, but that retention of activity may be influenced greatly by environmental conditions, virus strains, and perhaps other factors. Conflicting results have been reported by different investigators as to the importance of tobacco mosaic virus, carried as a surface con-

SEED TRANSMISSION OF PLANT VIRUSES

223

taminant, in the production of infection in seedlings. Doolittle and Beecher (1937), in some of the first tests with this virus, state that it is often found on the surface of seeds of diseased tomato plants and that, when such seeds are planted almost immediately after extraction, an occasional case of seed transmission had been noted. Effects of seed treatments on the amount of tobacco mosaic virus retained by tomato seed give further evidence indicating that the virus may occur on the surface of seeds, sometimes in considerable concentrations, and produce infection. Chamberlain and Fry (1950) compared uncleaned, fermented, and acid-extracted seed with respect to virus content and seed transmission and found that virus was transmitted by uncleaned seed, but not by seed extracted by fermentation or by acid. They suggested that virus was carried on the seed surface and that seedlings became infected during or shortly after emergence. Alexander (1960) failed to recover tobacco mosaic virus from infected mature tomato fruits extracted with hydrochloric acid and no infection was iound in 900 seedlings from fermented seed. Taylor et al. (1961) state that tobacco mosaic virus occurs both in and on tomato seeds from infected fruits and that seed-coat virus was eliminated by acid extraction and by trisodium phosphate treatment, but not by washing with detergents. No infection occurred in seedlings grown from 14-month-old seeds or from seeds that had been extracted with acid. It was concluded that loosely held virus on the surface of freshly extracted seeds is most likely to result in infection of seedlings and that any treatment that removes most or all of the superficial virus will reduce or prevent seed transmission of tobacco mosaic virus in tomato.

B. Virus Carried in Parts of Seed outside the Embrgo It is probable that a considerable number of viruses may be present in seeds in some stage of their development, even though they may not be seed transmitted. I n the process of seed development, quantities of carbohydrates are moved into the seed as a food reserve. Since there is evidence that virus movement in the phloem is correlated with carbohydrate transport, viruses that occur in high concentrations in the phloem would be expected to move in considerable quantities into seeds that have a vascular connection with the mother plant where they would accumulate as food reserves are increased. I n fact, movement through the phloem, in certain cases, might be more effective in introducing certain types of viruses into seed tissue than movement through the usual avenues of invasion present in various kinds of parenchyma tissue.

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c. w. BENNETT

Probably viruses that would be expected to occur in highest concentration in phloem are those that produce disturbances that arise primarily in the vascular system causing leaf curling, yellowing without mottling, witches’-broom, rosette, etc. Few such viruses have been investigated as to their occurrence in seed, but it has been shown that sugarbeet curly top virus occurs in relatively high concentration in the perisperm of seeds of infected sugarbeet plants (Bennett and Esau, 1936). Embryos separated from the remainder of the seed after beginning germination in a moist chamber contained no virus, whereas the remainder of the seed contained a high virus content. Other viruses of this general type may occur in seed of infected plants. However, no virus of this type has been shown to be seed transmitted. They apparently are unable to infect embryos, probably due to limitations in ability to extensively invade certain types of tissue. Several viruses that readily invade parenchyma tissue are reported to occur in parts of the seed outside the embryo. Sheffield (1941) obtained evidence from the study of inclusion bodies that severe etch virus infects the testa, but not the endosperm or embryo of seeds of Hyocyamus niger. In tests with the Lincoln and Virginia varieties of cowpea, Crowley (1959) found that, in plants infected with bean southern mosaic virus before blossoming, the virus was present in nearly 100% of the testae and endosperms of seeds of both varieties, but could not be detected in the embryos. By seed dissection and inoculation of suitable host plants, Crowley (1957) demonstrated the presence of bean common mosaic virus in the testa and embryo of bean seeds, tomato spotted wilt virus in the testa of cineraria and tomato seeds, bean yellow mosaic virus in the testa of bean seeds, and cucumber mosaic virus in the testa of cucumber seeds and in the testa and endosperm of seeds of wild cucumber. Seed transmission, however, was obtained only with bean common mosaic virus carried in the embryo. Gold et al. (1954) state that the concentration of particles, presumed to be virus particles, in the endosperm of seeds of barley plants infected with barley stripe mosaic virus was approximately as high as in leaf tissue. This virus also invades the embryo and is seed transmitted. Tobacco mosaic virus commonly occurs in the seed coat of tomato seeds (Chamberlain and Fry, 1950; Crowley, 1957, 1959; Taylor et al., 1961; Broadbent, 1965). Crowley (1957) found tobacco mosaic virus in the testae of seeds of pungent pepper, a plant in which McKinney (1952) had previously reported 21.6% seed transmission of a latent strain of tobacco mosaic virus in transplanted seedlings. Taylor et al. (1961) and Broadbent (1965) reported that tobacco mosaic virus occurs in a small percentage of endosperms of seeds of infected tomato plants, but they recovered no virus from embryos.

SEED TRANSMISSION OF PLANT VIRUSES

225

According to Ford (19661, pea streak virus occurs in immature seeds of infected pea plants, but is inactivated as the seeds mature. When immature seeds were germinated the virus was transmitted to 8 of 27 seedlings; no transmission was obtained through mature seeds. Assay results indicated that the virus was associated only with the seed coat and did not infect the embryo. Gilmer and Wilks ( 1967) have recently reported a surprisingly high percentage of seed transmission of tobacco mosaic virus in seeds of Malus platycarpa, and apple and pear. I n seeds of M . platycarpa the virus was present in excised inner seed coat-endosperm tissue, but it was not recovered from cotyledons or embryos of dormant seed. However, virus was recovered from 3 of 10 embryos and from 5 of 10 seed coats of dormant seeds of two varieties of apple. Virus titer in dormant seeds was low, but increased considerably during germination. The manner in which seed-borne viruses that do not occur in the embryo are transmitted to seedlings is not yet entirely clear in all instances. The higher percentages of seedling infection with tobacco mosaic virus in tomato, and also in pepper, have been reported when seedlings were transplanted. A few workers have reported seed transmission of tobacco mosaic virus to nontransplanted tomato seedlings (Milbrath, 1937; Chamberlain and Fry, 1950; Raychaudhuri, 1952). However, Milbrath (1937) found such transmission only with the variety Indiana Canner. Other investigators have reported no seed transmission of tobacco mosaic virus in thousands of seedlings grown from seed of infected plants (Caldwell, 1934; Jones and Burnett, 1936; Nitzany, 1960). For example, Jones and Burnett (1935) reported no seed transmission to any of 3368 plants grown from seeds of infected plants until after they had been handled by workmen. I n extensive investigations of seed transmission of tobacco mosaic virus in tomato, Taylor et al. (1961) and Broadbent (1965) found no seed transmission unless the seedlings were transplanted. Taylor et al. (1961) concluded that in the varieties with which they worked, seedlings grown from infected seedlings were contaminated, but not infected. Contamination was common on roots, but occurred on cotyledons only when the seed coats were elevated during germination. Infection occurred from the virus contaminating the roots and cotyledons during transplanting and could be extensive enough to be an important source of inoculum. However, Gilmer and Wilks (1967) report that, unlike tomato, infection of apple and pear seedlings with tobacco mosaic virus appear to be direct, since seedlings were infected prior to transplanting or handling. Invasion of apple and pear seeds appeared to be more extensive than invasion of tomato seeds. Tobacco mosaic virus was recovered from

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C. W. BENNETT

the embryos of a few seeds of apple, but not from embryos of seeds of

M . platycarpa in which, nevertheless, 38% seedling infection was reported. It would appear, therefore, that the virus would not be ex-

tensively embryo transmitted and that it may be able to infect seedlings in the early stages of their development in the absence of abrasions produced in handling. A relationship of virus to seed tissues outside the embryo that could bring about this result has not been clearly defined. 111. TRANSMISSION OF VIRUSESCARRIED IN

THE

EMBRYO

The number of viruses known to infect the embryo of seeds, and thus to be seed transmitted, and the number of plant species known to be involved in seed transmission of viruses have increased markedly in recent years. Crowley (1957) listed about 20 viruses as being seed transmitted in about 40 species of plants. More recently, Fulton (1964) listed 36 viruses transmitted through 63 species of plants. These numbers have been still further increased by more recent investigations. An attempt has been made in Table I to assemble available data on viruses and plant species involved in seed transmission, including reported percentage of seed transmission in each virus-host combination involved.

A. Methods of Embryo Infection Seed transmission of viruses, with few possible exceptions, is dependent on infection of the embryo in some stage of its initiation or development. Embryo infection probably can occur in any one of three ways: (1) through introduction of virus into the embryo sac by the male gametophyte, (2) through ovule invasion by virus from the mother plant, and (3) possibly occasionally through direct invasion of the embryo in some stage of its development following embryo initiation. 1. Infection through the Agency of Pollen

Early in the history of plant virus investigations, Reddick and Stewart (1918) suggested that bean common mosaic virus might be carried in pollen and that it might pass from the germ tube into the style in cross-pollination and produce infection. Reddick (1931) later found that when flowers of healthy bean plants were pollinated from infected plants, some of the resulting seeds transmitted virus, thus proving that pollen may carry virus and transmit it to the embryo. A short time later, Nelson and Down (1933), in studies of seed transmission of bean common mosaic virus in crosses between Refugee and Early Prolific varieties of navy pea bean, found that if only one

227

SEED TRANSMISSION O F PLANT VIRUSES

TABLE I TRANSMISSION OF VIRUSESTHROUGH SEEDS Disease induced by virus involved

Abutilon mosaic Apricot gummosis Arabia mosaic

Arabia mosaic Avocado sun-blotch Barley stripe mosaic’

Bean common mosaic Bean southern mosaic Bean western mosaic Bean yellow mosaic Beet 41 yellowsb Cherry leaf roll Cherry leaf roll Cherry necrotic ringspot

Cherry yellows Citrus psorosis Citrus psorosis (crinkly-leaf type) Citrus xyloporosis Clover (white) mosaic Clover yellow mosaic Coffee ringspot Cowpea mosaic Cucumber mosaic

Plant tested

Percent virus transmission

Reference

Abutilon thompsonii X 0.7 Keur (1934) A . mulleri Fridlund (1966) Prunua avium 15 Beta vulgaris 13 Lister and Murant (1967) Capsella bursa-pastoris 33 Lister and Murant (1967) Chenopodium album 80 Lister and Murant (1967) “Fragaria X ananassa” Lister and Murant (1967) 6.9 Glycine max Lister (1960) 6.3 Lactuca saliva 60-100 Walkey (1967) 1.2-25 Lamium amplezicaule Lister and Murant (1967) Lycopersicon esculentum 1.8 Lister and Murant (1967) Myosotis amensis 19-95 Lister and Murant (1967) Petunia hybrids 20 Lister (1960) 5.4-28 Plantago major Lister and Murant (1967) 21-100 Lister and Murant (1967) Polygonum persicaria 2.2 Lister and Murant (1967) Senecio vulgaris Stellaria media 57 Lister and Murant (1967) Perseu amcricana 76 Wallace and Drake (1953) Avena saliva McKinney (1965) 0-9.5 Commelina communis Inouye (lfJ62) ? Hordeum vulgare McKinney (1951.4 58 6.7-81 MoNeal and Afanasiev (1955) Triticum aestivum Phaseolus vulgaris Reddick and Stewart (1918) 50 Vigna sesquipedulis Snyder (1942) 37 Vigna sinensis Shepherd and Fulton (1962) 3-4 Skotland and Burke (1961) Phaseolua vulgaris 2-3 Lupinus lutew Corbett (1958) 6.2 Bet0 vulgaris 47 Clinch and Loughnane (1948) Glvcine max Lister and Murant (1967) 100 I’haseolua vulgaris 1240 Lister and Murant (1967) Viola tricolor Lister and Murant (1967) 1.2-6.1 Das et al. (1961) Cucurbita mazimu 2.7 Undetermined Hobart (1956) Prunua americanu 6.0 Prunus avium Cochran (1946) Prvnus cerasus 30 Cation (1949) Prunus mahaleb 10 Cation (1949) 3.6 Cochran (1950) Prunus pcrsica Prrnua cerasus 9.0 Cation (1952) Prunus mahaleb 8.7 Cation (1949) Childs and Johnson (1966) Citrw sinensis X Poncirrus 19 trijoliata Citrus s p . Trace Wallace (1957) CitTW Bp.

Trijolium pratense Trifolium p d e n s e Coffea excelsa Vigna catjang Vigna sinensis Vigna sinensis Cucvmis melo Cucumis sativus Echinoeyslis lobata Lupinus luteus vigna aesquipedalir Vigna ainensis

(Continued)

66 6.0 7.6 8.7 17 23 0-55 2.1 Trace 9.1 ? ? 4-28

Childe (1956) Hampton (1963) Hampton (1963) Reyes (1961) Capoor and Varma (1956) Capoor and Varma (1956) Anderson (1957) Kendrick (1934) Doolittle (1920) Doolittle and Gilbert (1919) Troll (1957) Anderson (1957) Anderson (1957)

228

C.

W. BEl”ETT

TABLE I-Continued Disease induced by

virus involved

Datura queroina Dodder latent mosaic Dodder latent monaio Elm m m i o Grape fanleaf Grape yellow mosaic Hop chlorosis Lettuoe monaio Lychnia ringspotC

Yuakmelon moaaic

Pea early browning Pea seed-borne mosaicd Peaah necrotic leaf spot Peanut marginal chlorosis Peanut mottle Peanut stunt Prune dwarf Raspberry buahy dwarf Raspberry ringspot Raspberry ringspot

Sowbane mosaio

Soybean monaio Squash monaio

Sugarcane moeaic Tobacoo mosaio* Tobaoco rattle

Tobacco ringspot

Plant tested

Datum atrarnmium om^

cuscutn calif

Cuscutn cumpastria Ulmus ametieam

Chenopodium amaranticolor Chenopodiumamaranticolor Humulw lupulus m u c o sativa Lactuco aerriolo Beta vulgaris Capaella bursa-pastoria Ceroetium &maurn Lyhnia divark-ata Silew gallico Silsw noctijlora Clleumia melo Clleurbito jlexuoaus Cucurbito moachata Cucurbita pep0 Piaum aativum Piaum sativum Prunus peraico Arachia hypogaea Arachia hypogaeu Arachia hppqaea Prunus maaus Rubw Mheua Beta vulgaris Capaella buraa-pastmia “Fragaria X a m m m ” Qlyoina ma2 Rubw idrreus Stellaria media Atriples pacijka Chenopodium album Chopodium murals Chenopodium quinoa Cnyiw maz Cucurbilo mazima cucurbito d o Cumubita mizta clleurbita pep0 Zea maya Malus platwarpa Mdus aylvestris P#rus oommunia Capaella buraa-past& Lamium amplszicoule Yyaaotia amensis Papaw r h Cucumia melo Cnucins m50 Loctuco satiw Nicotiom t a h u m Petunia hybrido Taradcum o&inala

Percent virus transmission 79

2.4 4.9 1.M.S 48 1.3 0.7 27 3.1 0.2-6.2 9.6 9.4 27 68 28 41 12-93 “Readily” “Readily” “Readily” 37 10-30 3-9 30-100 2 0.2 16 40-60 60-66

2.2-3.3 36 7.2 18 29 21 30 46 1.6 1-18 0.2-1.6 6.520 0.3 2.2 0.4 38 37 36 1.9 2.2 6.0 1.1 3-7 64-78 3.0 4.9 19 8-38

Reference Blakdee (1921) Bennett (1944s) Bennett (1944s) Breto (1960) Callahan (1957) Diaa (l963) Diaa (1963) Salmon and Ware (193s) Newhall (1923) van Hoof (1969) Bennett (1969) Bennett (1969) Bennett (1969) Bennett (1969) Bennett (l9S9) Bennett (1969) Rader et al. (1947) Rader et d. (1947) Rader el al. (1947) R d e r et d. (1947) BOBand van der Want (1962) Inouye (1967) Wagnon et al. (1960) van Veleen (1961) Kuhn (1966) Troutman et al. (1967) G i e r and Way (1960) Cadman (1965) Lister and Murant (1967) Lister and Murant (1967) Lister (19M) Lister (1960) Lister and Murant (1967) Lister and Murant (1967) Bennett and Costa (1961) Bennett and Costa (1961) Bennett and Costa (1961) Banoroft and Tolin (1967) Rose (1963) Grogan at al. (1969) Grogan et d. (1969) Grogan el d.(1969) Middleton (1944) Shepherd and Holdeman (1966) Gilmer and Wilke (1967) G i e r and wilka (1967) Gilmer and Wilks (1967) Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967) L ~ andr Murant (1967) YcLean (1962) Daajardins et d.(1964) Grogan and Schnathorst (1915) Valleau (1932) Henderson (1931) Tuite (1960)

229

SEED TRANSMISSION OF PLANT VIRUSES

TABLE I-Continued Disease induced by virus involved Tomato black ring

Plant tested Beta vulga& Capsello bursa-pmtm’s Cetostium vulgafum Chnopodium album “Fragaria X anunassa” Fumario ofiianalia Olucina maz

Lamium amplezicaule Liouahwm vulgare Lu~opersiemaeacukntum Mumotis anmmis Nicotioncr rusfica Po0 annua Pdugmaum peraimria Rubw idoeua Senecio nulgaria Spergtllo amensis stdlorio meah Tomato bunchy top Tomato r k p o t

vig7la ShW?l& Phuealis peruuiana

Sdanum incanum

(nucine mas

Percent virus transmission 66 90 33-100 84 40 100 83 10-48 5.7-8.3 19 100 4.4-8.8 2.7 21-100

1-6 14 63

66 23 29

63 76

Reference Lister and Murant Lister (1960) Lister and Murant Lister and Murant Liater (1960) Lister and Murant Lister (1960) L i t e r and Murant Lister and Murant Lister and Murant Lister and Murant Lister and Murant L i t e r and Murant Liater and Murant G t e r and Murant Lister (1960) Lister and Murant Lister and Murant Lister (1960)

(1967) (1967) (1967) (1967) (1967) (1967) (1967) (1967) (1967) (1967) (1967) (1967) (1967) (1967)

McCleau (1948) MaClean (1948) Kahn (1966)

Inouye (1962) and Niteany and Gerechter (1962) reported seed transmiasion of barley stripe mosaic virus in m number of species of grasses, but presented no data on percent seed transmission. Not clearly demonstrated to he caused by a virus. Causal virus similar to and distantly related serologically to barley stripe mosaic virus (Gibbs ef al.,

1963).

Both positive and negative results were obtained in tests to determine a relationship with this and two strains of bean yellow mosaic virus. The extent of paasage of the virus through the embryo in the three species liited is not known.

‘

parent was infected, about 25% of the F1 progeny carried the virus, regardless of which parent supplied the virus, indicating about equal effectiveness in transmission of virus through pollen and ovules in the varieties tested. However, Medina and Grogan (1961) have shown in extensive tests that, although relatively high percentages of seed transmission may be obtained through either pollen or ovule, the amount of transmission through either parental source may vary greatly depending on the variety of plant used. Other viruses that have been shown to be pollen transmitted are listed in Table 11, along with available data on percent transmission by both pollen and ovules. Gold et al. (1954) found rod-shaped particles associated with barley stripe mosaic infection in different tissues of barley plants, including pistils and pollen. Approximately 10% of the seedlings from seeds of healthy plants pollinated from infected plants showed symptoms of disease. Transmission through seeds of infected plants has been considerably higher than this, often 50% or more (McKinney, 1951a,b), so

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C. W. BENNETT

it may be that this virus is transmitted less efficiently through pollen than through ovules. However, pollen transmission of barley stripe mosaic virus evidently varies considerably with variety and different conditions, since Inouye (1962) reported up to 35% seed transmission through use of pollen from infected plants. Elm mosaic virus, which is transmitted through a relatively high percentage of seeds of the American elm (Ulmus americanu) , is transmitted through both pollen and ovules (Callahan, 1957). When pollen from infected plants was used to pollinate healthy plants, 30.5% of TABLE I1 TRANSMISSION OF VIRUSESTHROUGH POLLEN AND OVULES Percent virus transmission through

Disease induced by virus involved

Plant tested

Barley stripe moeaia Barley stripe moeaio Bean common mosaic Bean common mosaic Bean southern mosaic Cherry necrotic ringspot Elm mosaic Lettuce mosaic Lychnis ringspot

Hmdeum oulgare Hmdeum vulgare Phaseolus oulgaria Phaasdus vulgaris Phasedus vdgaris Cwurbita mazima Prunua cem8us U l m w arnwhna Laetwa saliva Luclrnia divarimla Silens noctiflora Prunw cermw Fragwia virgiiniana R d u s ap. R d w up.

Prune dwarf Rsspberry ringspot

Tomato black ring

Pollen 10.0 35.0 25.0° 18-76' 21.3 0.0 27.8 30.6 0.48 18.6 18.6 19.3 5.6 6.2 12.9

Ovule

Pollen and ovule

-

-

25.0" 8-76'

50.0" 21-86'

0.8

-

-

-

-

0.5

76.0' 5.8 30.7 28.7

48.0 5.2 36.6 37.6

10.5

44.0

3.2

10.3

-

ia.5

-

-

Reference

Gold et al. (1954) Inouye (1962) Nelson and Down (1933) Medina and Grogan (1961) Crowley (1959) Daa el al. (1961) Way and Gilmer (1958) Callahan (1957) Ryder (1964) Bennett (1959) Bennett (1969) Gilmer and Way (1960) Lister and Murant (1967) Lister and Murant (1967) Lister and Murant (1967)

'Only Range of results with two strains of bean common mosaic virus on three susceptible varieties of bean. four plants involved. a Approximate.

the resulting seeds carried the virus; when both parents were infected, 48% of the seeds were infected. The results with pollen from healthy

plants on infected plants were too limited to have statistical significance, although 3 of 4 seedlings from such a cross were infected. Lychnis ringspot virus was transmitted readily through pollen of the dioecious species, Lychnis divaricata and Silene noctiflora (Bennett, 1959). I n L. divaricata, when only the male plant was infected, 18.6% seed transmission was obtained; when only the female plant was infected, 30.7% of the seeds transmitted; and when both parents were infected, 33.6% of the seeds transmitted. Similar results were obtained with L. noctiflora, although there was a somewhat higher percentage of seed transmission when both parents were infected.

SEED TRANSMISSION OF PLANT VIRUSES

23 1

Ryder (1964), utilizing male sterility in testing the relative amount of pollen and ovule transmission of lettuce mosaic virus in lettuce, found only about one tenth as much transmission through pollen as through ovules. Within the past few years, it has been shown that some of the seed-transmitted viruses of stone fruits are pollen transmitted. Way and Gilmer (1958) found that 5 of 18 sour cherry seedlings from seeds of a cross between the varieties English Morello (healthy female parent) and Montmorency (infected male parent), were infected with necrotic ringspot virus. The same investigators (Gilmer and Way, 1960) found that two viruses may be transferred through pollen, either separately or in combination. Pollen was transferred from three sour cherry trees, infected with necrotic ringspot and prune dwarf viruses, to healthy English Morello plants growing in a greenhouse. Of the 88 seedlings obtained, 8 had only necrotic ringspot virus, 10 had only prune dwarf virus, and 7 had both viruses. It seems likely that most seed-transmitted viruses may be able to invade pollen, although actual transmission through the agency of pollen has been demonstrated in a relatively low percentage of seed-transmitted viruses, as shown in Table 11. Most viruses that are seed transmitted are transmissible also by mechanical inoculation. Some viruses have been recovered from pollen extracts. Rader et al. (1947) reported th a t seed-borne melon mosaic virus was recovered from pollen, but apparently no tests for pollen transmission were made. Ehlers and Moore (1957) collected pollen from Shiro plum and Early Richmond, Montmorency, and Dyehouse varieties of sour cherry trees known to be infected with various stone fruit viruses. Inoculum was prepared by grinding pollen in phosphate buffer after which the mixture was introduced into suitable index plants. Viruses designated A, B, and E were transmitted from pollen. Since the identity of the viruses was not determined, it is not known whether they are seed transmitted. Also, Williams and Smith (1967) recovered virus from pollen of pear trees with pear decline symptoms but the virus was not identified and is not known to be seed transmitted. Limited information is available regarding the ability of noiiseedtransmitted viruses to invade pollen. Bennett and Costa (1961) recovered sowbane mosaic virus from pollen of Atriplez coulteri, but found no seed transmission in this species, although the virus was transmitted tlirough the seeds of other species of Atriplez. Also, Cadman (1965) found that pollen from plants of Chenopodium quiiioa and C. nmamizticolor, infected with apple clilorotic leaf spot virus (raspberry bushy dwarf virus) from raspberry, Malus, Prunus, and Anthriscus

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plants, contained sufficient virus, when macerated and used as inoculum, to produce 1 to 10 lesions per inoculated leaf of test plants, although no evidence was obtained that this virus is seed transmitted in either of the species of Chenopodium. Apparently, virus content of pollen of plants infected with viruses that are not seed transmitted has received very little attention. It may well be that pollen of many plants is highly resistant or immune to virus invasion, even in case of highly invasive viruses such as tobacco mosaic virus. More extensive tests of virus content of pollen of diseased plants would be of interest. Although transmission of virus to the female gametophyte through the medium of pollen has been known for nearly 40 years, it is only recently that information has been obtained as to whether virus so introduced is restricted to the ovule and its derivatives or whether it can escape and infect the mother plant. The possibility of transmission of bean common mosaic virus from diseased to healthy plants through the medium of pollen has been suggested (Reddick and Stewart, 1918; Reddick, 1931), but transmission of this type in bean has not been demonstrated. No transmission of Lychnis ringspot virus was found in plants of Lychnis divaricata, pollinated from diseased plants and pruned back to induce new growth, although more than 30% of the seeds of these plants carried virus, indicating that, even though virus was carried to the ovule by pollen, it did not escape from the flowers and systemically infect the mother plant (Bennett, 1959). Similarly, Lister and Murant (1967) found no evidence that healthy mother strawberry plants became infected when their flowers were pollinated from plants infected with raspberry ringspot virus which is seed transmitted in strawberry. Gilmer and Way (1960) transferred pollen from three sour cherry trees, infected with prune dwarf and cherry necrotic ringspot viruses, to healthy English Morello plants in the greenhouse. Thorough indexing of 50 of these plants after seed was harvested, indicated that all were virus-free, although virus was transmitted to about 25% of the seeds produced on the trees. Despite the earlier results which failed to show transmission of viruses to mother plants through infected pollen, other tests have given evidence that such transmission may take place, although it may occur only rarely. Way and Gilmer (1958) suggested that a t least a part of the field spread of necrotic ringspot virus of cherry may result from infection of trees through the agency of pollen from infected trees in the same orchard. In tests conducted under greenhouse conditions to assure freedom from accidental infection, Das and Milbrath (1961) transferred pollen from

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plants of Buttercup squash (Cucurbita maxima), infected with the Oregon RS 31 strain of stone fruit ringspot virus, to healthy squash plants. Of the 97 pollinated plants, 9 showed symptoms of disease. Twenty-nine flowers were pollinated on the 9 plants that became infected. It is pointed out that, since normally 100 or more ovules are fertilized in each squash fruit, and since only 9 of 97 pollinated plants were infected, the percentage of pollen grains that transmitted virus to the mother plant in these tests was extremely low. It is suggested further that, since a cherry fruit develops from a single ovule, demonstration of pollen transmission of this virus from one cherry tree to another by means of pollen would be extremely difficult. However, considering the large number of blossoms produced annually by a single cherry tree, it is proposed that a very low percentage of pollen transmission to trees could account for the natural spread of ringspot virus noted in cherry orchards. It is of interest in this connection that Gilmer and Way (1963) have since presented evidence indicating that pollination of both sour and sweet cherry from trees infected with cherry yellows virus may result in infection of the pollinated trees. Working in Canada, George and Davidson (1963) reported additional evidence of transmission of both cherry necrotic ringspot and cherry yellows viruses from tree to tree through the agency of pollen, and Cadman (1965) reported transmission of raspberry bushy dwarf virus to healthy plants of the Lloyd George variety of raspberry under greenhouse conditions by pollen from infected plants. Since it now seems evident that it is possible for virus to escape from an infected ovule and invade the mother plant, one may wonder what mechanism prevents this from occurring more often. It may well be that viruses do escape from ovules much more frequently than has been demonstrated, but are unable to invade adjacent tissues at s a c i e n t l y rapid rates to permit virus to become established in the mother plant and produce systemic infection. The rapid rates of virus movement usually occur in the phloem and there is evidence that this movement is correlated with carbohydrate transport. Since carbohydrates would be expected to move more or less unidirectionally toward the fruit, this avenue for rapid invasion of the mother plant from the ovule would be largely eliminated, even if virus could reach the phloem. Invasion of tissues outside the phloem might be limited to the relatively slow movement of virus from one parenchyma cell to another. It would be expected that movement of a virus from a cherry ovule, for example, through the surrounding tissue by this method and down through the fruit pedicel into the fruit spur, wholly through parenchyma tissue, would require considerable time. I n most cases, fruits might mature

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and be harvested before this occiirs, even if the virus should move out of the ovule. 9. Infection by Ovule Invasion by Virus from the Mother Plant It is relatively simple to determine the presence or absence of a number of viruses in the male gametophyte of the plant by direct tests, but comparable determinations are dif3icult or impossible in the female gametophyte. The evidence as to direct invasion of ovules by virus from the mother plant, therefore, is largely circumstantial. Despite this fact, evidence indicating such invasion is reasonably extensive and convincing. It seems logical that a virus that can invade male gametes should also be able to invade female gametes. Moreover, in cases of pollen transmission, virus is undoubtedly introduced directly into the embryo sac from the pollen tube during the process of fertilization where it may persist and infect the embryo. Thus, if the embryo sac can be infected through direct introduction of virus by pollen, it seems likely that ovules may be infected through virus invasion from adjacent cells of the mother plant in the early stages of ovule development, or later. Other evidence for ovule infection is to be found in the correlation between seed transmission and time of infection of the mother plant. Fajardo (1928) reported that bean plants grown from seeds of plants infected with bean mosaic virus gave higher percentages of infected seeds than plants inoculated during stages of vegetative development and that there was no virus transmission by seeds of pods set prior to infection of the mother plant. This has been confirmed by other work (Fajardo, 1930; Nelson, 1932; Harrison, 1935). Nelson concluded that seed transmission of bean mosaic virus depends on the ability of the virus to reach the ovule before or shortly after fertilization. Also, Couch (1955) found that lettuce plants, inoculated with lettuce mosaic virus just before flowering, produced fewer virus-infected seeds than plants infected soon after planting. Plants that became infected after flowering did not transmit the virus through seeds. Crowley (1957) obtained further evidence of ovule infection by growing bean plants a t two temperature levels, only one of which permitted seed transmission of bean common mosaic virus. I n these tests, only temperatures to which the plants were subjected prior to fertilization of flowers influenced seed transmission, indicating a definite relationship between seed transmission and ovule infection. Somewhat similar results were obtained in temperature-reversal tests with barley stripe mosaic virus in barley (Singh et al., 1960). It may be concluded, therefore, from the evidence thus far available,

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that viruses that are seed transmitted are capable of infecting ovules. I n fact, it seems probable that, in the great majority of cases, ovule or embryo sac infection, by one means or another, is essential for seed transmission. Even in the few cases in which this may not be true, such infection apparently greatly increases seed transmission. 3. Infection through Direct Invasion of the Embryo Although evidence indicates that, as a rule, seed transmission is dependent on infection of the embryo in a very early stage of its development by virus that has first invaded the embryo sac, some evidence indicates that this may not necessarily be true in all cases. Although Hagborg (1954) found that wheat plants inoculated with barley stripe mosaic virus a t the time of heading gave no seed transmission, Eslick and Afanasiev (1955) found markedly different results with this virus in barley. Field plants, inoculated at 10-day intervals from the 1-leaf stage through the hard-dough stage, all gave seed transmission. In the susceptible variety Compana, seed transmission increased up to and including the boot stage, in which stage seed transmission reached 63.7% ; it then declined but remained relatively the same within the range of 10.1-14.7% transmission from heading to hard-dough stage, inclusive. I n the more resistant variety Titan, seed transmission tended to increase up to and including the boot stage, where it reached 4.4%, but there was no evidence that later inoculations of the mother plant resulted in seed transmission. The results of Eslick and Afanasiev (1955) were confirmed in part by Crowley (1959), who concluded that infection of young developing barley embryos with barley stripe mosaic virus apparently is possible, although high percentages of infection occurred only when plants were inoculated before flowering. Also, in carefully controlled experiments, Crowley (1959) found that bean southern mosaic virus, which invades seeds, but is later inactivated and not seed transmitted, was able to infect bean embryos 4 days, but not 7 and 10 days, after flowering. However, barley stripe mosaic virus was not detected in embryos of Lincoln and Virginia varieties of soybean from plants inoculated after flowering, although the virus was present in both testa and endosperm tissues of both varieties. It was concluded, also, that a small percentage of embryos of the Lincoln and Chippewa varieties of soybean may become infected with tobacco ringspot virus in early stages of development. These results indicate that in certain plants some viruses are able to invade developing embryos, possibly without first having invaded ovules or embryo sac. In fact, in the case of transmission of barley stripe mosaic virus in barley seeds, it would appear from some results

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that invasion of the embryo may be possible, even after it has reached a stage of development approaching maturity (hard-dough stage of the seed). This, however, requires corroboration before it can be fully accepted, since the tests in which these results were obtained were performed with field plants and there is no proof that seed transmission of virus in the later dates of inoculation were not associated with delayed flowering in some of the test plants.

B. Types of Viruses That Are Seed Transmitted As may be noted by referring to Table I, seed-transmitted viruses have certain general characteristics more or less in common. Most are readily juice transmissible to one or more host plants, indicating an ability to invade parenchyma tissue. The diseases they induce are, for the most part, characterized by disturbances that arise in parenchyma tissues. Symptoms, therefore, consist chiefly of mottling, local necrotic or chlorotic lesions, etch, and other types of abnormalities that have their origin mainly in parenchymatous tissues. Several of these viruses, especially the ringspot viruses, induce initial acute effects from which the plants later recover, sometimes almost completely. Viruses that give evidence of causing symptoms that arise primarily in the phloem, or other parts of the vascular system, appear not to be seed transmitted. This applied to viruses that produce such symptoms as leaf curling, rosetting, witches1-broom, and general yellowing in the absence of mottling or other disturbances arising primarily in parenchyma tissue. Viruses transmitted by certain types of vectors appear to be more often seed transmitted than those transmitted by other types of vectors. For example, no leafhopper-transmitted viruses are known to be seed transmitted. This applies also to aphid-transmitted viruses that are persistent in their vectors. Several nonpersistent aphid-transmitted viruses, however, are seed transmitted. Perhaps the most marked association between vector type and seed transmission is found with nematode-transmitted viruses, most of which have been shown to be seed transmitted. Some of these viruses are transmitted through seeds of a considerable number of host plants and, in a number of cases, to high percentages of seeds of infected plants (Table I). This association is so marked that Lister and Murant (1967) have suggested that seed transmission seems to be highly characteristic of nematode-transmitted viruses.

C. Influence of Environmental Factors on Seed Transmission The effect on seed transmission of environmental factors that influence plant growth and development has received relatively little attention. Studies that have been made, however, suggest that percent-

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age of seed transmission of some viruses may be markedly influenced by the environment under which seeds are produced. Of the environmental factors that may influence seed transmission, temperature appears to be the most important of those thus far investigated. Crowley (1957) reported that seed transmission of bean common mosaic virus ranged from 0 to 25% when infected bean plants were grown a t two temperature levels. No seed transmission was obtained in seeds of plants grown at 62" to 65"F, whereas 16 to 25% seed transmission was obtained from seeds produced at 68°F. Different results, however, were obtained with this virus by Medina and Grogan (19611, who found that bean plants grown continuously a t 60" to 66°F gave 67.4% seed transmission, whereas plants grown continuously a t 80" to 90°F gave 56.7%seed transmission. The reason for this marked difference in results with the same virus is difficult to explain on the basis of available evidence, but Medina and Grogan (1961) suggest that it most likely resulted from their use of a bean variety (Sutter Pink) with a higher propensity for seed transmission, although other factors may have been involved. Results somewhat parallel to those obtained by Crowley for bean common mosaic virus were reported by Singh et al. (1960) in studies of the effect of temperature on seed transmission of barley stripe mosaic virus in four tolerant varieties of barley. They found 3% seed transmission in one variety and no seed transmission in the remaining three varieties when plants were grown a t 16"C, whereas seed transmission in the same four varieties ranged from 9 to 27% in seeds of plants grown at 20°C and from 7 to 28% in seeds of plants grown at 24°C. In contrast with the evidence of increased seed transmission of bean common mosaic and barley stripe mosaic viruses in seeds of plants grown at higher temperatures, Crowley (1959) found a depressing effect from higher temperatures in seed infection by bean southern mosaic virus in soybean. In a test in which infected plants were grown at two different temperatures, 95% embryo infection occurred in plants grown a t 16" to 20"C, whereas only 55% infection occurred in plants grown at 28" to 30°C. This difference in percentage embryo infection was not found to be associated with a difference in virus concentration of leaf samples taken from these plants a t flowering time. Further studies of the effects of temperature on seed transmission would be of value in determining the reasons for the variations in seed infection and transmission observed in plants grown a t different temperature levels. There is evidence that percentage seed transmission may be correlated to some degree with severity of symptoms. Severity of symptoms in turn may be correlated with virus concentration (Pound,

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1952; Bancroft and Pound, 1956; Singh et al., 1960). Low virus concentrations may result from slow rates of virus increase. This might be accompanied by slower rates of invasion of cells of developing tissue at growing points, which might permit developing gametes to escape infection. However, this is wholly speculative and much more evidence will be required before definite conclusions may be reached.

D . Influence of Host Plant and Virus o n Seed Transmission A considerable amount of information dealing with the influence of host plant and virus on seed transmission has become available from recent as well as from earlier work. Lister and Murant (1967) list plant species in which nematodetransmitted viruses were found to be seed transmitted. These include many distantly related or unrelated species, ranging in one case (tomato black ring virus) from a representative of the family Gramineae to a representative of the family Compositae, and including 13 families. Arabic mosaic virus was transmitted through seeds of 9 families. Different viruses of the group were transmitted through different percentages of seeds of the same species. For example, tomato black ring virus was transmitted to 83% of the progeny of Capsella bursa-pastoris, but to only 2% of the progeny of the Malling Exploit variety of raspberry ; whereas raspberry ringspot virus was transmitted to only 3% of the progeny of C. bursa-pastoris, but to 18% of the progeny of the Malling Exploit variety of raspberry. These two viruses were transmitted independently, and in different percentages, through seeds of Malling Exploit raspberry plants infected with both viruses. Many other seed-transmitted viruses, however, are carried through seeds of a more limited number of species, as may be seen by referring to the list of seed-transmitted viruses in Table I. Apparently some viruses may be seed transmitted in one or a few species and not so transmitted in a number of other susceptible species. Tobacco mosaic virus appears to be an interesting example of this type. Tests of seeds of tobacco, tomato, pepper, and many other susceptible species have indicated complete lack of seed transmission of this virus, although it may be seed-borne and produce infection of seedlings a t the time of, or shortly after, seed germination. Recently, however, Gilmer and Wilks (1967) reported seed transmission of tobacco mosaic virus through seeds of Malus platycarpa and through seeds of varieties of apple and pear. Since the virus was recovered from the embryo of dormant seeds of apple, it would appear that it can be embryo transmitted in such seeds. Restricted seed transmission is known also with a number of other

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viruses. Dodder latent mosaic virus was transmitted through the seeds of Cuscuta californica and C . campestris, but not through the seeds of cantaloupe, buckwheat, and pokeweed, all of which are susceptible to systemic infection (Bennett, 1944a)b). Species of related genera have shown marked differences in seed transmission of viruses. In some of the very early work, Doolittle and Gilbert (1919) found 10% transmission of cucumber mosaic virus through seeds of wild cucumber, Echinocystis lobata, but little or no transmission through seeds of cultivated cucumber. Grogan et al. (1959) found percentages of seed transmission of squash mosaic virus in different types of cucurbits ranging from 0 to 20.7%) and apparently barley stripe mosaic virus is transmitted through a much higher percentage of seeds of barley and wheat than through seeds of oat (McKinney, 1965). Recently, Childs and Johnson (1966) reported transmission of citrus psorosis virus through from 15 to 31% of the seeds of “Carrizo” citrange. More extensive tests are needed t o determine whether this virus is transmitted through other kinds of citrus, but the fact that extensive tests have given no evidence of seed transmission in the common varieties of citrus suggests that seed transmission of psorosis virus may be highly restricted in citrus types. Species of the same genus have also shown marked differences in seed transmission of viruses. For example, sowbane mosaic virus was transmitted through 21.4% of the seeds of Atriplex pacifica, but not through seeds of five other species of this genus (Bennett and Costa, 1961), and coffee ringspot virus was transmitted through 8.7% of the seeds of Coffea excelsa, but through none of the seeds of C. arabica (Reyes, 1961). Marked differences in seed transmission also have been found in different varieties of the same species. Some of these differences are as great as those between unrelated species. Grogan et al. (1959) found no seed transmission of squash mosaic virus through seeds of Early Summer Golden Crookneck squash, but the virus was transmitted through 5.1% of the seeds of Zucchina squash. Ross (1963) reported 11.1% seed transmission of soybean mosaic virus through seeds of soybean variety Lee, and less than 1% transmission through seeds of the variety Hill, and Kennedy and Cooper (1967) found 20.6% seed transmission of this virus in seeds of the variety Harosoy and no seed transmission in the variety Merit. Mottling of seeds was associated to some degree with percentage of seed transmission, but it was not definitive. McKinney ( 1965) reported transmission of barley stripe mosaic virus in up to 9.5% of the seeds of some varieties of oat and p o seed transmission in Certain other varieties, Tobacco ringspot virus

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(strain 98) was transmitted through approximately 3% of Paris Island Cos variety of lettuce but not through seeds of the Imperial 615 variety (Grogan and Schnathorst, 1955). Lettuce mosaic virus was transmitted through from 1 to 3% of the seeds of lettuce varieties commonly grown in California, and through up to 8% of the seeds of the variety Bibb (Grogan et aZ., 1952), but it was not transmitted through seeds of the variety Cheshunt Early Giant (Kassanis, 1947; Broadbent et aZ., 1951; Couch, 1955). Eslick and Afanasiev (1955) found that when barley plants were inoculated with barley stripe mosaic virus in the boot stage, 64.7% of the seeds of the variety Compana were infected, whereas only 4.4% of the seeds of the variety Titan were infected. This difference occurred despite the fact that both varieties seemed to be equally susceptible. Other evidence, however, indicates that percentage seed transmission may in some cases be more or less correlated with degree of severity of symptoms of disease on the mother plant. Tests of one susceptible and four tolerant varieties of barley gave less than 15% seed transmission of barley stripe mosaic virus in seeds of the tolerant varieties and up to 75% transmission in seeds of the susceptible variety (Singh et uZ., 1960). In tests of 10 varieties of barley, graded 1 to 5 on the basis of apparent resistance and inoculated with barley stripe mosaic virus on seven dates from March 7 to May 10, inclusive, Inouye (1962) found a marked correlation between resistance and percent seed transmission. In the most susceptible variety, for example, seed transmission ranged from 26 to 100% in the 7 inoculations, whereas in two of the most resistant varieties, no seed transmission was obtained from any date of inoculation. Other evidence indicating that resistance of a variety may affect seed transmission, was reported by Smith and Hewitt (1938), who collected seed from 118 field-inoculated selections of bean, representing 51 varieties, and tested the seed for transmission of bean common mosaic virus. The seeds of the 51 varieties were divided into four classes on the basis of severity of induced symptoms. Results indicated a correlation between symptom severity and percentage of seed transmission. The average seed transmission a t Berkeley, California, for classes 1 to 4, respectively, was 6.8, 9.4, 20.3, and 36.1%; corresponding values at Davis, California, were 1.1,20.8, 23.3, and 30.4%. According to Troutman et al. (19671, seed transmission of peanut stunt virus varied with the severity of the disease on the plant from which the seeds were harvested and with seed size, and Kuhn (1965) states that transmission of the peanut mottle virus appeared to occur more frequently in smaller seeds and in discolored seeds.

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In studies of the influence of resistance of the parent on seed transmission of bean common mosaic virus, Medina and Grogan (1961) presented conclusive evidence that the degree and type (dominant or recessive) of resistance of the parent may markedly influence seed transmission. In their tests, the F1 progeny resulting from pollination of resistant plants with pollen from infected susceptible plants, contained a large number of infected plants when resistance was recessive and no infected plants when resistance was dominant. Medina and Grogan suggest that the simplest explanation for the lack of seed transmission to the F1 progenies of resistant varieties in which resistance is dominant is that dominance of resistance precludes seed transmission. This, in effect, would be a type of immunity to infection of the embryo. The F1progeny of varieties with the recessive type of resistance would lack this immunity. Obviously, effects of this kind could give highly contradictory results in studies of the effect of resistance on seed transmission, if only severity of symptoms on the parent plants were used as a measure of resistance. Possible differences in the amount of seed transmission of different strains of the same virus have been studied in only a few instances, but available evidence indicates that strains of some viruses may differ in the readiness with which they are transmitted through seeds. This type of evidence may become even more impressive after relationships of some of the reported seed-transmitted viruses, such as those affecting legumes, are more clearly defined. Grogan and Schnathorst (1955) reported that “strain 98” of tobacco ringspot virus was transmitted through approximately 3% of the seeds of Paris Island Cos Lettuce, whereas a “calico” virus, believed to be a strain of tobacco ringspot virus, was not seed transmitted in this variety, thus indicating a difference in seed transmission between two strains of the same virus. Extensive evidence of an effect of virus strain on seed transmission has been presented by McKinney (1965), who investigated the relationship of a large number of strains of barley stripe mosaic virus on varieties of barley, wheat, and oat. This virus is normally transmitted through high percentages of seeds of diseased plants of barley and wheat. In tests of seven strains on varieties of barley and wheat, for instance, McKinney found average seed transmission ranging from 36.8 to 53.0%. However, with a number of other strains, seed transmission ranged down to a low level and a few strains were not seed transmitted under the conditions of the tests. Further evidence of a relationship between virus strain and seed transmission was found in oat, in which seed transmission ranged from 0 to 9.5% ; at least two of seven strains tested were not seed transmitted in the varieties of oat used.

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In summary, it may be said that investigations of the effects of host plant and virus on seed transmission of viruses indicate a marked influence by both host and virus, without providing any very definite information on the basic factors involved in the extreme variations in seed transmission that have been observed to occur as a result of the influence of the host, the virus, or the interactions of host and virus. Some of the results, however, do suggest a possible correlation between resistance and seed transmission, thus opening the distinct possibility that seed transmission may be controlled by genetic factors that can be utilized in breeding programs designed to reduce or eliminate seed transmission of viruses in certain crop plants. Success in such a program could be of great value in the control of virus diseases, such as lettuce mosaic and barley stripe mosaic in which seed transmission is an important factor in disease initiation and spread.

E . Longevity of Viruses in Seeds The length of time viruses retain their activity in seeds appears to vary with the virus involved, the host plant, the tissue in which the virus occurs, and perhaps other factors, as indicated by the results shown in Table 111. Some viruses are lost in a short time, whereas others appear to retain activity as long as the seeds remain viable. However, as yet no pattern of survival of viruses in seeds has emerged. The speed with which virus activity is lost in some seeds is marked. The virus of bean southern mosaic, which occurs in high concentrations in the young embryo and other parts of the bean seed, becomes inactive in the embryo, usually by the time the seed is mature, and for this reason is not normally seed transmitted (Cheo, 1955). Sugarbeet curly top virus, which may occur in relatively high concentrations in sugarbeet seeds, but which is not seed transmitted, was active after 2 months, but not after 3 months, in seeds stored a t room temperatures. This virus is concentrated in the perisperm of the beet seed (Bennett and Esau, 1936). It is known to retain activity for as long as 8 years in dry beet leaves (Bennett, 19421, and the reason for its short period of survival in the seed is unknown. Tobacco mosaic virus, which may occur in the testa and endosperm of tomato seeds, but not in the embryo, is inactivated slowly during storage (Taylor e t al., 1961). Middleton and Bohn (1953) report that seed transmission of muskmelon mosaic virus dropped from 95% in fresh seed to 5% in 3-year-old seeds, but virus was still recoverable from seeds stored 5 years. Middleton (1944) found no difference, however, in the percentage of seed transmission of squash mosaic virus in squash seeds tested shortly after harvest and 3 years later,

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Valleau (1939) found a lower percentage of tobacco seeds carrying tobacco ringspot virus after 5.5 years of storage. He pointed out, however, that differential loss of viability might account for the results. A marked decrease in percentage of seeds transmitting muskmelon mosaic virus after 3-years' storage was reported by Rader e t al. (1947). Fulton (1964) found that the percentage of Prunus pensylvanica seeds that TABLE 111 LONQEVITY OF VIRUSESI N SEEDS Disease induced by virus involved Barley stripe mosaic Bean common mosaic Bean southern mosaic Bean western mosaic Cherry necrotic ringspot Dodder latent mosaic Hop infectious sterility Lychnis ringspot Muskmelon mosaic Raspberry ringspot Snwbane mosaic Squash mosaic Sugarbeet curly top Tobacco mosaic Tobacco ringspot Tomato black ring Tomato bunchy top

Plant tested Hmdeum uulgare Pheolua vulgaris 0Z~lcin.esoja Phaaeolua vulgaris Phaaeolua vulgaris l'runua pemulwnica Cuacvtcl campa9tris Humulua lupvlua LWhnis divaricata Silene gallica Cucumis me10 Cucumis melo Capsella bursa-pastoris Stellaria media Chenopodium murale Cucurbita pep0 Beta adgaris Lucopersicon esculentum Nieotiana tabacum Petunia violacea Capsella bursa-pastoris Stellaria media Phusalis wruvitrna Solanum incanum

Period virus retained activity in seeds (years) 6% 3 2 <7 months 3 6 1

2 992 days" 843 days 3 6 6 6 G%b

3 2 months" 9d 656

I months 6

6

1 year, 2 months 344

Reference Scott (1961) Nelson (1932) Kendrick and Gardner (1924) Zaumeyer and Harter (1943) Skotland and Burke (1961) Fulton (1964) Bennett (1944b) Blattny and Osvald (1964) Bennett (1969) Bennett (1969) Rader et al. (1947) Middleton and Bohn (1953) Lister and Murant (1967) Bennett and Costa (1981) Middleton (1944) Bennett and Esau (1936) Broadbent (1965) Valleau (1939) Henderson (1931) Lister and Murant (1967) Lieter and Murant (1967) McClean (1948) McClesn (1948)

Later tests showed virus activity after 9 years.

* Later teats showed virus activity after 14 years. Not seed transmissible. Not embryo transmitted.

transmitted cherry necrotic ringspot virus remained relatively constant a t 60-70% the first 4 years of storage a t 2"C, but dropped to less than 5% by the sixth year. In this case there was little loss in seed viability, indicating a true loss of active virus in the seed. With several other viruses there is no evidence of loss of virus activity in seeds with time of storage. Nelson (1932) found about the same amount of seed transmission of bean common mosaic virus in seeds of navy pea bean in fresh seeds as in seeds 3 years old. H e states that it has been shown that this virus probably can survive as long as seeds remain

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viable. The percentage seed transmission of bean western mosaic virus was not reduced after 3 years of storage (Skotland and Burke, 1961). Lychnis ringspot virus showed no reduction in percent transmission through seeds of Lychnis divaricata after 992 days (Bennett, 1959). Later tests showed no decrease in percentage transmission after 9 years, although seed germination was reduced by about 90%. Tests with sowbane mosaic virus in seeds of Chenopodium murale showed no diminution of percent seed transmission after 6.5 years (Bennett and Costa, 1961). Subsequent tests showed no reduction in percent seed transmission after 14 years. Scott (1961) found that barley seeds stored 6% years transmitted barley stripe mosaic virus to 65% of the seedlings. This is approximately the percent of seed transmission found by McKinney (1951b), who first reported seed transmission of this virus. Lister and Murant (1967) found that raspberry ringspot and tomato black ring viruses were active in seeds of Capsella bursa-pastoris and Stellaria media after 6 years of storage. It seems probable, from the results available, that there are several viruses that may remain active in seeds as long as the seeds are viable. The relationship of virus content of seeds to viability apparently has not been investigated, but there is no conclusive evidence that viability of seeds is affected by the presence of virus, although such effects are possible. Apparently, no tests of virus content of seeds that have become nonviable due to age have been made. It may be that in some cases, virus activity extends beyond the normal span of life of infected embryos. Since most of the seed-transmitted viruses are juice transmissible, such tests could be made readily. Also, in the case of some viruses, such as barley stripe mosaic virus, which Scott (1961) has shown to be detectable readily by serological means, tests could be made easily, although it is possible that retention of virus activity would not necessarily be proven by positive serological tests. Retention of active virus in seeds provides an effective and economical method of preserving some of the seed-transmitted viruses, as pointed out by Wilson and Dean (1964). The value of desiccated tissues in preserving viruses has been indicated (Bennett, 1942; McKinney, 1947, 1965). It is well known, also, that viruses may retain activity indefinitely when stored at temperatures below freezing. Williams and Smith (19671, for example, recovered virus from pollen of a pear tree with pear decline symptoms after the pollen was air dried and held at -18°C for 5.6 years. However, where they can be utilized for this purpose, seeds have obvious advantages in preserving viruses for future use, especially where infected seeds retain their viability for long periods.

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F. Effect of Seed-Transmitted Virus on Seedlings Perhaps one of the most characteristic features associated with seed transmission of viruses is the ability of embryos, infected in the very early stages of formation, to survive, grow, and mature into plants that usually are productive and often almost normal. However, the degree to which young seedlings show symptoms varies considerably with different viruses and different host plants. I n some cases, symptoms are evident from the very early stages of growth. Symptoms of bean common mosaic usually are evident on the first pair of true leaves (Nelson, 1932). Symptoms of sowbane mosaic are evident on the cotyledons of plants of Chenopodium murale before they are more than half full size (Bennett and Costa, 1961), as are symptoms of Lychnis ringspot on cotyledons of Lychnis divaricata and Beta vulgaris (Bennett, 1959). Ring patterns, caused by Lychnis ringspot virus, are sometimes marked on cotyledons, especially on the relatively large cotyledons of seedlings sugarbeet plants. Cochran (1946, 1950) found a host difference in relation to production of symptoms on seedlings by Prunus ringspot virus, which is transmitted through low percentages of seeds of Mazeard cherry and Love11 and Rio Oso varieties of peach. Ring and mottle symptoms were produced on Mazzard cherry seedlings, but peach seedlings were symtomless. Callahan (1957) found that elm seedlings from seeds carrying elm mosaic virus, as determined by indexing on Havana 38 tobacco plants, might or might not show symptoms. The factors involved in this result, however, were not determined. McKinney (1953) states that barley plants manually inoculated with barley stripe mosaic virus first develop one or two leaves that are almost a solid ivory color, denoting an acute phase of the disease; plants then recover and show only a milder or chronic phase of disease. When the virus is transmitted through the seed, however, seedlings show only mild symptoms characteristic of the chronic phase of the disease. Symptom production was influenced by temperature at which seedlings were grown and also by light intensity. Tests with virus-infected seeds of the Glacier variety of barley showed that temperatures of 75" to 100"F, with adequate light, favored the production of the characteristic mildly and moderately chlorotic markings in the first three leaves of the seedlings. I n tests in the greenhouse during winter months it was necessary to use supplementary light for production of best symptoms (McKinney, 1954). Wallace (1958) and Wallace and Drake (1953, 1962) found that avocado trees may be infected with avocado sun-blotch virus, but show no symptoms of disease. Seedlings from seeds of such trees were symp-

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tomless, but a high percentage carried virus that produced sun-blotch symptoms when graft-transmitted to virus-free seedlings. Different results were obtained, however, with trees that showed sun-blotch symptoms. Most seedlings from trees with symptoms were virus-free, but occasional seedlings did become infected through seed transmission of the virus. These seedlings, unlike those from seeds from symptomless trees, showed characteristic symptoms of sun-blotch. Valleau (1932) states that seedlings from seeds of tobacco plants infected with tobacco ringspot virus are likely to be symptomless unless grown a t low temperatures, under which conditions they might be dwarfed and show some yellowing of leaf margins, but no ringspots typical of the disease on juice-inoculated plants. Somewhat similar results were obtained by Desjardins et al. (1954) with tobacco ringspot virus in soybean. Seedlings from infected seeds showed marked symptoms when grown a t low temperatures, but tended to be symptomless a t higher temperatures. Henderson ( 1931) states that petunia seedlings from seeds infected with tobacco ringspot virus were dwarfed and stunted, but soon outgrew the disease. Recently, Lister and Murant (1967) reported extensive observations on the effect of seed-transmitted virus on seedlings of plants infected with nematode-transmitted viruses. Most of the tobacco rattle virus-infected seedlings showed necrotic markings on some leaves and were dwarfed. However, with other viruses of this group, one of the most outstanding features of seed transmission was the predominantly healthy appearance of infected seedlings. Often they were indistinguishable from noninfected seedlings of the same batch except by virus assay. When groups of raspberry ringspot infected and noninfected seedlings of Malling Exploit variety of raspberry and Huxley variety of strawberry, together with tobacco black ring infected and noninfected seedlings of Redgauntlet variety of strawberry and their various virus-infected plant clones were grown in a cool greenhouse for 2 years, plants of the parent clones regularly showed symptoms during the spring and late summer flushes of growth but the seedlings remained symptomless. There were, however, exceptions to the rule that infected seedlings appeared healthy, particularly with progenies of infected Capsella bursapastoris plants. Infected seedlings were symptomless when grown in summer but seedlings grown in winter were yellow. The yellowing, however, was general and unlike the ring-and-line patterns which developed in manually inoculated seedlings. Leaves of yellowed plants, produced later, looked normal and growth was not noticeably reduced. However, after seeds from healthy and infected plants were stored in cellophane bags a t room temperature for 6 years, seedlings from infected seeds were considerably smaller than those from noninfected

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seeds. No such difference in vigor, however, was observed in Stellaria media seedlings from 6-year-old seeds. In general, it may be concluded that seedlings from virus-infected seed are likely to show symptoms that are similar to those shown by the mother plant in a chronic stage of disease. If there is no recovery in the mother plant, seedlings are likely to show symptoms as severe as those of the mother plant; if the disease is characterized by the production of an acute stage in the mother plant, followed by a chronic stage, the seedlings tend to show symptoms of the chronic stage; if the mother plant recovers to a high degree, seedlings tend to be symptomless. It appears, therefore, that the recovery stage of a virus disease is usually carried through the seed.

G . Economic Importance of Seed T r a n s r n k w n of Viruses For many years relatively little economic importance was attached to seed transmission of viruses. Few viruses were known to be seed transmitted and the economic effects of seed transmission in these cases were considered to be limited largely to local spread of disease in annual crops. Accumulated evidence has now shown, however, that nearly 50 viruses, with about 120 virus-host relationships, are involved in seed transmission. Some of the seed-transmitted viruses are wholly or partially dependent on seed transmission for widespread local and long-distance dissemination. Among these viruses are some that are capable of causing severe damage to the crop plants which they attack. Doolittle and Gilbert (1919) were among the first to attribute economic importance to seed transmission of a virus when they pointed out that a certain percentage of the seeds of wild cucumber plants (Echinocistis lobata), infected with cucumber mosaic virus, may carry virus and establish centers of infection from which virus may be carried to commercial cucumber fields by natural vectors. Fajardo (1930) emphasized the importance of seed transmission in carrying over bean mosaic virus from one season to the next and its role in the widespread dissemination of bean mosaic through movement of commercial seed lots. Seed transmission of this virus is recognized as an important factor in bean production in many areas where susceptible varieties of bean are grown commercially. It is probable that seed transmission is one of the chief factors in the extensive geographical distribution of bean mosaic. Transmission of barley stripe mosaic virus through seeds of diseased plants of barley and wheat is responsible for significant losses to these two crop plants in a number of grain-producing areas of the world. According to Slykhuis (1967), the perpetuation of this disease in both barley and wheat is dependent on seed transmission of the causal virus. Seed-borne virus has been reported by several investigators (see

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Taylor et al., 1961) as important in initial infection and later spread of tobacco mosaic virus in tomato. Such transmission results from infection of seedlings at the time of seed germination, largely from surfaceborne virus much of which can be eliminated by proper seed treatment or handling. Taylor et aZ. (1961) have shown, however, that tobacco mosaic virus may occur in a small percentage of endosperms where it may have greater protection against the usual types of seed treatment. They consider seed-borne tobacco mosaic virus an important source of infection in tomato. Seed transmission of some viruses may be very important in virus spread even when only a small percentage of seeds carry virus. For example, lettuce mosaic virus is rarely transmitted through more than 5% of seeds of diseased plants, but seed transmission is indicated as one of the major factors in the spread of lettuce mosaic in England (Broadbent et al., 1951), and Grogan et aZ. (1952) determined that its spread in California is largely a local phenomenon and that the most important initial virus source in lettuce fields is infected seed. Mosaicfree seed is an effective means of controlling the disease in large-scale field plantings (Zink e t al., 1956). The significance of even very low percentages of seed transmission in the spread of lettuce mosaic is emphasized by the finding that if percentage of seed transmission exceeds 0.1% the degree of control is likely to be unsatisfactory. The use of virus-free seed is now one of the major methods of control of lettuce mosaic in California and Arizona. The development and use of lettuce varieties in which lettuce mosaic virus is not seed transmitted would probably reduce lettuce mosaic from a disease of major importance to one of relatively minor economic consideration. In some cases, where seed transmission may be of little or no importance on the immediate crop, it may still have importance in the transport and establishment of viruses over long distances. Reyes (1961), for example, states that there probably has been considerable spread of coffee ringspot virus in the Philippine Islands through the use of seeds of diseased plants of Coflea excelsa. Sugarcane mosaic virus, according to Shepherd and Holdeman (1965), has spread more rapidly in corn, and other susceptible species, than is readily accounted for by aphid transmission. It is possible that a small percentage of seed transmission in corn has enabled this virus to become established in localities far removed from the area of original distribution. Childs (1956) states that citrus xyloporosis symptoms noted in Israel on unbudded sweet lime seedlings could be explained on the basis of the evidence that xyloporosis virus is transmissible through seeds. Seed transmission of viruses has presented added difficulties to the

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production of virus-free nursery stock in stone fruits and avocado and it may prove to be important in other tree fruits such as citrus. For example, cherry necrotic ringspot virus is now known to be seed transmitted and rather widely distributed in varieties and species of Prunus, in some of which its presence is difficult or impossible to detect from visual symptoms. Gilmer (1955) reported that Prunus maha2eb seeds imported from France for use as rootstock material showed from 5.5 to 16% seedling infection with a virus that appeared to be cherry necrotic ringspot virus when indexed on cucumber. Gilmer further states that J. S. Boyle found as high as 40% seed infection with ringspot virus in seeds of St. Medard cherry. Use of virus-infected material of this type to supply rootstocks for nursery trees could lead to considerable loss in orchard plantings. Wallace and Drake (1962) state that the use of seed from avocado trees infected with sun-blotch virus in the production of rootstock material has been responsible a t times for high percentages of disease in commercial nurseries. Cadman (1963) suggested that seed transmission probably plays an important role in the survival and spread of some of the nematodetransmitted viruses. Murant and Lister (1967) found that virus-free populations of vector nematodes can acquire tomato black ring virus, raspberry ringspot virus, and arabis mosaic virus from weed seedlings from infected seeds. When soil containing infective nematodes (Longidorus elongatus) were left fallow, infective nematodes retained tomato black ring and raspberry ringspot viruses for only about 9 weeks, but when weed seeds in the fallowed soil were allowed to germinate, nematodes reacquired virus from infected seedlings. Thus, virus may be retained in infected seeds and serve as a source of inoculum, presumably as long as infected seeds remain viable. Murant and Lister (1967) point out that instead of major transport of virus by vectors, which occurs with most viruses, the situation is somewhat reversed with nematode-transmitted viruses in that virus dissemination may take place to a much greater extent through distribution of infected seed than through vectors. They state that seed dissemination of nematode-transmitted viruses could explain the fact that these viruses, despite having vectors that seem unable to transport them over a distance, are widespread, even though their distribution may be scattered and localized, Distribution through seeds is further indicated by the finding of plants infected with tomato black ring virus and raspberry ringspot virus in areas free of the vector nematode. It is suggested that probably these two viruses have reached a large proportion of the populations of L. elongatus in eastern Scotland through seed transport. Although obviously any virus that is seed transmitted has a potential

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for being transported long distances in seed, nematode-transmitted viruses would appear to have greater potential for this type of dissemination because of the wide range of plant species in which they are seed transmitted and the high percentage of seeds of diseased plants that may carry virus. I n some cases of very high percentages of seed transmission, these viruses woudd be expected to persist several generations, even in the absence of a vector, which might add to their chance of becoming established in any area where a vector exists.

INFLUENCING OR DETERMINING SEEDTRANSMISSION IV. FACTORS Early in the history of plant virus research Allard (1915), in work with tobacco mosaic virus, pointed out that a very efficient barrier guards against infection of the embryo of seeds and prevents passage of virus to seedlings. With the growth of the science of virology the high degree of protection afforded young sporophytes in seeds against invasion by viruses present in the mother plant has become even more evident, despite the fact that an appreciable number of viruses are now known to be seed transmitted. The factors involved in preventing passage of virus through the seed to the next generation have received attention by a number of investigators. I n general, factors suggested have included ( a ) inactivation of virus in the embryo, ( b ) sterility of infected gametes, thus preventing production of infected seed, and (c) inability of virus to infect young embryos due either to resistance of the embryo to infection or inability of virus to infect male and female gametes prior to embryo initiation. It has been pointed out, also, that any virus that is restricted to the vascular system could not be seed transmitted, since there are no vascular connections between the embryo and the mother plant. This may be the explanation of the fact that, thus far, seed transmission has been confined to viruses that give evidence of being able to invade parenchyma tissue extensively.

A. Inactivation of Virus in the Embryo The possibility that inhibitors may affect seed transmission of a virus probably was first proposed by Duggar (1930). On the basis of inhibitory effects of seed extracts on infection with tobacco mosaic virus, he suggested that specific proteins, or other specific substances present in the seed, might prevent seed transmission of tobacco mosaic virus. According to Kausche (1940), tobacco mosaic virus may be present in unripe seeds, and sometimes in dried seeds, but is inactivated by a substance elaborated during ripening and germinating processes. More recently, Caldwell (1962) suggested that the embryo may be an unfavorable medium for virus increase because of a low concentration of high-

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energy phosphntcs requircd for virus multiplication. However, if low concentrations of such compounds do widely influence seed transmission, it would seem that more evidence of virus inactivation in the developmental stages of the embryo should be available and that seed transmission should not show the sharp correlation with introduction of virus a t the time of fertilization which is evident in so many instances. Although there has been no confirmation of inactivation of viruses in embryos by specific inactivators, evidence has been obtained that indicates strongly that certain viruses may be inactivated even after they have invaded the embryo. Gold et al. (1954), in studies of rod-shaped particles in embryos of barley seed from plants infected with barley stripe mosaic virus, found that particles occurred in all of the seeds of diseased plants examined in about the same concentration as in leaf tissues. Since the seeds came from lots that showed only about 50% seed transmission, it was suggested that if the particles observed were the causal agents of the disease, one would have to postulate inactivation of the virus during seed storage to account for healthy seedlings from seeds of diseased plants. However, if inactivation of this virus does occur in the seed, it would seem more likely that it may take place in the development stages of the embryo rather than in storage, since Scott (1961) has shown that barley stripe mosaic virus is remarkably durable in barley seed and may produce as much as 64% infection of barley seedlings after 6.5 years of seed storage. Perhaps the best evidence for inactivation of a virus in the seed has been obtained with bean southern mosiac virus in bean. Zaumeyer and Harter (1943) recovered this virus from extracts of bean seeds in the milk and early dough stages and from newly ripened seeds, but failed to recover it from seeds that had been stored 7 months. I n more extensive tests, Cheo (1955) found that bean southern mosaic virus infects the young embryo and increases in concentration as the embryo matures. However, virus concentration dropped to a low level or reached zero as the seed dried. Seedlings from immature seeds that germinated on filter paper showed 58 to SO% infection, whereas seedlings obtained from mature seeds usually contained no virus. Extracts from mature or germinated seeds caused greater virus inhibition than extracts from immature seeds, but inhibition was never complete from any of the extracts used.

B . Sterility of Gametes Direct lethal effects of virus on gametes or embryos that might prevent production of infected seeds has been reported in a limited number of instances. Tobacco ringspot virus may cause pollen sterility and re-

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duce seed yield, but the virus apparently does not have a comparable effect on the ovule (Valleau, 1932). A somewhat similar condition obtains in seed transmission of lettuce mosaic virus in lettuce. This virus causes a high degree of pollen sterility and very little pollen transmission of the virus occurs, but ovule transmission is not affected to the same degree (Ryder, 1964). High percentages of pollen sterility were found in barley plants infected with barley stripe mosaic virus (Yamamoto, 1951; Inouye, 1962). Inouye reported that reduction in the number of fertilized seeds in wheat infected with this virus in Japan amounted to 20 to 50% but apparently there was no corresponding reduction in percentage of infected seeds. Almost complete pollen sterility in Daturu stramonium plants, infected with Datum quercina virus, was reported by Blakeslee (1921) , but seed transmission of the virus was obtained when diseased plants were pollinated from healthy plants. Couch (1955) suggests that lack of seed transmission of lettuce mosaic virus in the variety Cheshunt Early Giant may be due to a hypersensitivity of floral tissue to a high level of virus activity which results in the blighting of floral shoots. However, there is a recovery phase in this variety in which seeds may be produced that do not give infected seedlings. Calavan and Christiansen (1966) reported excessive seed abortion in several types of citrus infected with citrus stubborn disease virus. This virus is not known to be seed transmitted, but there is no evidence as to whether seed abortion is related to freedom from seed transmission. The relationship of this virus to pollen produced by infected plants would be of interest. A direct effect of tomato aspermy virus on both pollen and ovules of tomato has been described by Caldwell (1952) in which the virus interferes with the normal meiotic processes of both megaspore and microspore and prevents seed formation in diseased plants. It is suggested that interference by virus with nuclear division may be more widespread and of greater biological significance than is generally realized.

C. Susceptibility of Gametes to Infection Although it seems probable that in some cases other factors may influence seed transmission, the available evidence indicates that, in the great majority of instances where seed transmission occurg, it is associated with the ability of virus to infect developing embryos following invasion of the female gametophyte either directly from the mother plant or through introduction of virus by the male gametophyte. This conclusion seems justified on the basis of known relationships of pollina-

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tion to seed transmission, the associations that have been determined between time of infection of the mother plant and seed transmission, and certain other intluences that have already been mentioned. Anatomical, cytological, and other factors that may be associated with apparent freedom of the gametophytic generation of many plants from virus infection have received attention from a number of investigators including Esau (1938, 1948), Bennett (1940, 1956), Crowley (1957), Fulton (19641, Schneider (1965), Baker and Smith (19661, and Lister and Murant (1967). Obviously, freedom from infection by gametes produced by systemically infected plants must be due either to inherent resistance of gametes to virus invasion and increase or to their escape from infection through the operation of an effective protective mechanism. Whether the gametophytic generation of plants differs from the sporophytic generation with respect to resistance to viruses, or other pathogenic agents, is difficult to determine, but this possibility would appear to exist. If there are such differences, they would not be expected to result solely from the haploid nature of the gametophytes, since there is no evidence that ploidy, as such, affects resistance to plant viruses. Sugarbeet resistance to curly top, mosaic, or yellows, for example, does not appear to be different in haploid, diploid, triploid, or tetraploid plants of comparable lines of material. However, the metabolic effects of a reduction division that results in production of male and female gametophytes may be basically different from those involved in change in the chromosome numbers in the sporophyte. It might be of interest to make tests of relative resistance of sporophytio and gametophytic generations to plant pathogens in groups where the two generations are separate plants as in liverworts, mosses, and ferns. Despite the difficulty of making determinations of resistance of gametophytes to virus infection, there is evidence that in some instances the female gametophyte may be immune to virus infection, although the full significance of the results obtained thus far are not clear. According to Medina and Grogan (1961), when healthy plants of the bean varieties Red Mexican and Idaho Refugee, both having a dominant type of resistance to bean common mosaic virus, were pollinated from infected susceptible varieties, no seed transmission was obtained. Since pollen from infected susceptible plants transmitted to other susceptible plants, it seems evident that virus must have been introduced into the ovules or embryo sacs of the resistant plants by the infected pollen where it failed to infect the developing embryo. Further tests of virus content of pollen and seedlings of resistant plants might be of value in further clarification of these results.

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Although gametes or gametophytes may have some effect in inactivation of viruses, and thus in reducing or preventing seed transmission, it now seems probable that gametes may more often escape infection, possibly through inability of viruses to fully invade primary meristem. Virus invasion of growing points has been shown to be incomplete in a number of instances, as indicated by the isolation of virus-free tissue from growing points of virus-infected plants and the production of virusfree cultures from the excised tissue. Literature on this subject has been reviewed recently by Hollings (1965) and by Schneider (1965). Obviously, delay in the invasion of meristematic tissue by a virus might permit the initiation of virus-free gametes. The theory of relationship between early invasion of meristematic tissue and seed transmission is further supported by the fact that seed transmission is often associated with virus diseases from which infected plants partially or completely recover. Virus invasion of meristematic tissues and the establishment of a relationship between virus and cells in early stages of maturation has been proposed as the most probable explanation for recovery of plants from a severe or acute stage of infection and the development of a less severe or chronic stage of disease so evident with a number of virus diseases (Valleau, 1941; Benda and Naylor, 1957). A correlation between recovery from symptoms and seed transmission has been observed in a number of cases. Wallace (1958) and Wallace and Drake (1962), for example, report a high percentage of seed transmission of avocado sun-blotch virus in infected avocado trees that showed no symptoms, but a low percentage of seed transmission in trees with symptoms. Moreover, seedlings from seeds of symptomless trees were symptomless, whereas seedling from seeds of trees with symptoms showed marked sun-blotch symptoms. Further evidence on the relationship between symptoms and delayed invasion of meristem has been obtained by Lister and Murant (1967) in experiments in which strawberry plants, infected with a nematodetransmitted virus, were propagated from individual crown buds. All crown buds from plants infected from seed contained virus, whereas virus was recovered from only a portion of crown buds from naturally infected plants. Thus, presence or absence of virus in the growing points of infected plants seemed to be determined by whether or not the infected plants had recovered from the acute stage of the disease. Widespread seed transmission of several other nematode-transmitted viruses and an association of seed transmission with recovery of the mother plant and the passage of the recovery stage through the seeds of recovered plants, as reported by Lister and Murant (1967), lends

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additional support t o the theory of a relationship between seed transmission and ability of viruses to invade meristem. This relationship, however, does not yet offer a full explanation for some types of seed transmission. Low percentages of seed transmission in 33me plants recovered from certain viruses would indicate either incomplete invasion of the meristematic tissue of such plants or the intervention of additional factors that can prevent gamete infection. Also, there are relatively high percentages of seed transmission of some viruses, such as those causing bean common mosaic and sowbane mosaic, from which the mother plants do not recover and in which the infected seedlings show symptoms of the same degree of severity as the mother plant. Furthermore, not all viruses from which plants recover have been found to be seed transmitted. Beet curly top virus, from which both tobacco and tomato plants show high degrees of recovery, apparently is not seed transmitted in any of its host plants. However, this virus may well occur in meristematic tissue, since Lackey (1946) has reported recovery of virus from root-tip tissue beyond the vascular system in sugarbeet and from cambium of tobacco stems. Also, Benda and Bennett (1964) found that when young tobacco seedlings were inoculated with curly top virus as they emerged from the seed coat, they may become infected and show only the recovery stage of the disease. Similar results in the initiation of the recovery stage in small tomato plants in the absence of a chronic stage have been reported more recently (Benda and Bennett, 1967). On the basis of these results, curly top virus might be expected to be able to invade pollen and ovules. However, in tests by Bennett and Esau (19361, no virus was found in pollen of recovered tobacco plants. Curly top virus shows a close relationship with the phloem of infected plants and seems to be unable to invade certain types of parenchyma tissue such as medullary rays of the woody cylinder of tobacco and Nicotiana glauca (Bennett, 1934). If this virus is capable of invading meristematic tissue, as some of the evidence indicates, but does not survive in parenchyma cells, the logical conclusion is that it must become inactivated during the process of cell maturation. Whether inactivation of this nature is involved in the failure to detect virus in pollen of infected plants remains to be determined, but if such inactivation does occur it would be expected to influence, and perhaps prevent, seed transmission. Escape of the pollen mother cell from virus invasion through delayed invasion of meristem, and the subsequent escape of pollen grains from invasion through rapid development, is not d a c u l t to visuali~e,although it is not clear why there is the wide range in pollen infection

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in different host/virus combinations and why there is an apparent degree of uniformity in the percentage of pollen infection in some specific host/virus combinations. However, the mechanism which protects ovules from infection between the time of formation and the initiation of the embryo is more difficult to understand, especially now that it has been shown that virus introduced into the embryo sac through the agency of pollen is able, in some cases, to escape from the embryo sac and infect the mother plant. It would appear, on the basis of the respective periods that male and female gametes may be subject to invasion by virus from the mother plant, that much more transmission should occur through the female than through the male gametes. Although evidence is admittedly too fragmentary and conflicting to justify a conclusion, this does not necessarily appear to be true (Table 11).

D . Protection of the Embryo from Virus Infection Regardless of the mechanism which protects male and female gametes from infection, there must be an additional mechanism which can protect the embryo throughout its developmental stages from infection from neighboring cells, many of which in systemically infected plants may have a high virus content. In such cases, virus must either be unable to multiply and persist in the embryo after entrance or it must be excluded. Since, as already indicated, there is evidence of inactivation of viruses in the embryo in only rare instances, it seems likely that in the great majority of cases of lack of seed transmission the embryo escapes infection. If embryos are protected from virus invasion by exclusion throughout the course of their development, as appears probable in most instances of lack of seed transmission, the mechanism for exclusion must be relatively effective, but not necessarily very complicated. I n fact, it may consist merely of the interposition of structures between the mother plant and the embryo through which viruses are unable to move. When an embryo is initiated by union of nuclei in an uninfected embryo sac the embryo begins development in a virus-free medium. After cell walls are formed around the zygote there apparently are no protoplasmic connections between cells of the embryo and cells of adjacent tissue and the embryo becomes essentially a parasitic structure able to grow and develop through absorption of nutrients from the mother plant. In cases of freedom from virus invasion it is evident that the embryo can absorb essential food materials from areas containing both nutrients and virus without becoming infected. The most likely structure that could effect this separation would appear to be the cell walls of the embryo. There is evidence in only a very few cases that viruses are able to

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pass through the cellulose structure of cell walls. Kassanis et al. (1958) found that, although no plasmodesmata were observed connecting protoplasts of adjacent cells in tissue cultures, tobacco mosaic virus moved through tissue at a rate of about 1 mm per week. However, they report that, although cultures of callus tissue occasionally became infected when dilute suspensions of virus were poured over them, injuries were usually required, and that the number of infections depended on the type and number of injuries. Tissues infected through superficial injuries usually became virus-free after subculturing, whereas those infected by needle-prick remained infected permanently. It would appear, therefore, that even in tissue cultures, cell walls may offer some resistance to virus movement. Much evidence suggests that the cell wall, in the absence of penetrating cytoplasmic strands, usually is a formidable barrier to virus passage, Work by Caldwell (1932) indicates inability of tobacco common mosaic virus to move into living cells from intercellular spaces of normal leaves and inoculation of plants. Introduction of viruses into intercellular spaces by means of hypodermic needles, or the introduction of virus into tracheary elements through severed stems and petioles, has been very ineffective in producing infection. Schneider and Worley (1959), however, reported evidence indicating that bean southern mosaic virus can cause infection in intact living cells following transport in tracheary elements of a local-lesion host. In this instance, virus apparently was able to pass through cell walls of tracheae as well as through intact cell walls of adjacent parenchyma cells. However, even with this indicated ability to move through cell walls, the embryo of bean appears to have a high degree of protection against invasion, since, according to Crowley (1959), southern bean mosaic virus infected bean embryos 4 days, but not 7 and 10 days, after flowering. This indicates a rapid development by the embryo of resistance to invasion by a virus found by Cheo (1955) to multiply readily in the developmental stages of the embryo. As already indicated, barley stripe mosaic virus has been reported to be seed transmitted in barley plants inoculated up to and including the dough stage of seed development (Eslick and Afanasiev, 1955). Although the stage of development of individual embryos at the time of infection was not determined, it would appear that some infection may have occurred well after the beginning of embryo development. Working with the same virus, Crowley (1959) concluded that infection of young developing embryos is apparently possible in some varieties of barley; however, a high percentage of seed transmission occurred only when plants were infected before blossoming. Despite the fact that in rare cases viruses appear to be able to move

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through the cellulose structure of the cell wall, the best evidence regarding movement of viruses through parenchyma tissue still favors the theory that the usual path of movement is through plasmodesmata connecting protoplasts of neighboring cells. Any doubt as to whether viruses can move along plasmodesmata connecting adjacent cells seems to have been removed by the recent work of Esau et al. (19671,who found that in sugarbeet plants affected by beet yellows disease, virus particles were present in sieve elements, pores of sieve plates, plasmodesmata connecting sieve elements with parenchyma cells, and in plasmodesmata connecting parenchyma cells. Their observations indicated also that beet yellow virus can move from cell to cell in the form of complete particles.

E . Conclusions The weight of evidence available at the present time appears to indicate that transmission of viruses through the embryo may be influenced and conditioned by a number of factors and that no single factor can be used to explain all seed transmission or lack of it. However, it would appear that one of the determining factors involved in seed transmission of viruses, in the great majority of cases, is the ability of virus to infect male and female gametophytes. Whether this is determined by the ability of virus to invade meristematic tissue and thus enter the gametes in early stages of development, as much evidence indicates, or whether invasion is determined in part by susceptibility of the gametophytic generation to infection, is difficult to determine. I n m y event it appears also, except in rare instances, that embryos become immune to virus invasion shortly after their initiation, regardless of the concentration or the distribution of virus in the mother plant. Evidence indicates that this immunity may result from the absence of protoplasmic connections between the embryo and adjacent cells and from the inability of most viruses to move directly through the cellulose structure of the walls of cells that form the surface layer of the embryo.

REFERENCES Alexander, L. J. (1960). Phytoputhology 50, 627 (Abstr.). Allard, H. A. (1915). J . Agr. Res. 5,251. Anderson, C. W. (1957). Phytoputhology 47, 515 (Abstr.). Baker, I(.M., and Smith, 8.H. (1966). Ann. Rev. Phytopathol.4,311. Bancroft, J. B., and Pound, G. S.(1956). Virology 2,29. Bancroft, J. B., and Tolin,S. A. (1967). Phytopathology 57, 639. Benda, G. T.A., and Bennett, C. W. (1964). Virology 24,97. Benda, G. T.A., and Bennett, C. W. (1967). Am. J . Botany 54,1140. Benda, G. T. R., and Naylor, A. W. (1957). Am. J . Botanv 4.4, 443.

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