VIRUS ASSAY OF SEEDS FROM SELECTED MONTMORENCY CHERRY TREES'

VIRUS ASSAY OF SEEDS FROM SELECTED MONTMORENCY CHERRY TREES' J. A. GEORGE AND T. R. DAVIDSON Research Station, Canada Department of Agriculture, Vineland Station, Ontario Received March 17, 1966 ABSTRACT Assays for virus content were made on seed from 40 Montmorency sour cherry trees in an orchard where necrotic ring spot and sour cherry yellows viruses were spreading, the former very rapidly. Virus was detected in 3.5% of the 'sound' seeds from 10 virus-free trees, in 38.4% from 10 trees with shock symptoms, in 77.7% from 6 trees with sour cherry yellows symptoms, and in 93.1 % from 7 trees with necrotic ring spot symptoms. The number of seeds with virus, on otherwise virus-free trees, did not provide a reliable indication of the tendency of a tree to become infected. The average percentage of aborted seeds was: sour cherry yellows infected trees 35%, necrotic ring spot trees 22 %, trees in shock 28%, and non-infected trees 15%. INTRODUCTION Gilmer and Way (9, 10) showed that the necrotic ring spot and prune dwarf viruses could be transmitted by pollen from diseased Montmorency sour cherry trees to the seeds of fruits on healthy Montmorency trees. This was done by manual pollination of emasculated blooms on healthy trees with pollen from virus-infected trees. Although they obtained 20-25% diseased seedlings, none of the maternal trees became virus-infected. Later George and Davidson ( 6, 7) obtained evidence that this method could result in the subsequent infection of mother trees. Further work by Gilmer and \Vay (11) and Gilmer (8) substantiated these results. The tests reported here were conducted in 1961 prior to the first report of tree-to-tree transmission by pollen ( 6). Furthermore, it was not known what percentage of the seeds on healthy or diseased trees were infected under natural conditions. Therefore, tests were conducted to determine the numbers of virus-carrying seeds occurring naturally on virus-infected and non-infected trees in an orchard where natural virus spread was rapid, and to obtain an estimate of the numbers of diseased seeds on healthy trees that subsequently became infected. METHODS An 8-year-old Montmorency orchard was selected where both the necrotic ring spot virus (NRSV) and the sour cherry yellows virus (SCYV) were present and spreading. Annual spread of these viruses in this orchard has been shown diagrammatically as orchard 2 in a previous publication ( 4). In the spring of 1961, forty trees were selected for testing. At that time the virus history of these trees, based on symtomatology, and indexing on peach seedlings was: 10 virus-free; 10 infected with SCYV (some of these also carried recurrent NRSV); 10 infected with recurrent or latent NRSV; and 10 recently infected trees that showed 'shock' symptoms (3). From each tree a sample of 150 ripe fruits was picked at random from a height of about 5 ft around the tree. The fruits were stored at -15 C. During the winter of 1961-62, 50 'sound' seeds (i.e. those filling at least two-thirds of the pit cavity) were assayed for virus by trituration and mechanical inoculation to cucumber seedlings ( 5). These 'Publication No. 119. Can. J. Plant Sci. Vol. 46 (1966) 501

502 CAXADIAX JOURNAL OF PLAXT SCIENCE [Vol. 46 seedlings were then placed in a growth room under continuous light at 24 C. Symptoms were observed and recorded at 3 and 5 days from inoculation. Subsequent observation of the test trees to the end of 1965 provided further evidence of the virus content of the trees at the time the fruits were collected. For instance, two trees that were diagnosed in 1961 as infected with a nonrecurrent NRS virus later displayed SCY symptoms; and trees that were classed as having only SCY have since displayed recurrent NRS etch symptoms. Therefore, where pertinent, this more complete virus history was taken into consideration and the trees were regrouped accordingly. RESULTS AND DISCUSSION Test trees with similar virus histories, and symptoms, were grouped and the results, obtained from the virus assay of seeds from trees in each group, were compared (Table 1). Proportion of Virus-Infected Seeds Recurrent necrotic ring spot.-seven of the forty test trees persistently displayed symptoms typical of recurrent NRS. A consistently high average of 93 % (90-98%) of the 'sound' seeds of trees in this group carried detectable virus (Table 1). Such results indicate that only occasionally could virus-free seedlings result from trees with recurrent NRS symptoms. Recurrent sour cherry yellows.-the only virus symptoms to appear on six of the test trees were the characteristic yellow leaves of SCY that appeared for a brief period annually. Of the 'sound' seeds from trees in this group only 78% (54-94%) tested as virus-infected (Table 1). This was 15% lower than the average of the recurrent NRS group. Though some of the trees in the recurrent SCY group had as high a proportion of infected seeds as trees in the recurrent NRS group, many others had a much lower proportion. This may indicate an incomplete invasion of the floral parts by the SCYV and may in part account for the slower rate of field spread of SCYV as compared with NRSV ( 4). Necrotic ring spot and sour cherry yellows.-some of the 40 test trees showed symptoms of both viruses. When SCYV symptoms predominated, as indicated by consistently strong yellows symptoms with only mild etch, 80% of the seeds were infected. But when NRSV symptoms were predominant Table 1. The percentage of virus-infected seeds, and aborted seeds, from Montmorency sour cherry trees showing various virus symptoms Virus in 'sound' seeds Aborted seeds No. of Virus symptoms trees Av. (3) Range (3) Av. (3) Range(%) Recurrent NRS 7 93.1 90-98 22 11-31 Recurrent SCY 6 77. 7 54-94 35 29-46 SCY predominant + NRS 4 80.0 74-84 35 30-42 NRS predominant+ SCY 2 99.0 98-100 10 9-11 Unclassified 1 46.0 28 Shock in 1961 10 38.4 16-84 28 14-41 Shock in 1962 9 3.4 0-10 7-27 Shock in 1964 1 4.0 12 15 ABBREYIATIONS: NRS = necrotic ring spot; SCY = sour cherry yellows.

September 1966] GEORGE AND DAVIDSON-VIRVS ASSAY OF SEEDS 503 99% of the seeds carried virus. It would seem therefore that in some cases of dual infection the SCYV suppressed the number of seeds with detectable virus. Of the 20 trees diseased before 1960, whether they displayed symptoms of NRS, SCY, or both, only from one did all 50 seeds assay as infected. This tree displayed symptoms of both viruses with NRS symptoms predominant. Thus, in breeding trials, it may be possible to obtain a few virus-free seedling progeny from virus-infected parents. Shock.-Test trees that showed the shock symptoms of an initial infection in 1961 varied most widely in the percentage of 'sound' seeds with detectable virus ( 16-84 %, average 38 %). This wide range probably reflects the variation in degree of virus penetration throughout the newly infected tree. However, this variability was not associated with symptoms as all trees in this group were rated as having moderate to severe shock affecting two-thirds or more of each tree. Since their shock year ( 1961), 9 of these 10 trees have shown etch symptoms indicative of NRSV infection. The remaining tree has been symptomless. Unclassified.-One test tree indexed positive for virus when planted but remained symptomless for 10 years (1954-63). In 1964 the first symptoms appeared in the form of a moderate etch typical of NRS; in 1965 etch reappeared along with the first symptoms of SCY. The atypical symptom history and the exceptionally low percentage of seeds with detectable virus ( 46%) lead the authors to suspect that this tree contained an atypical combination of viruses or strains of viruses, with the propagation budwood and (or) rootstock as the probable sources. Detailed studies of the isolates were not made. No virus symptoms.-of the 40 test trees, 10 were symptomless and indexed as virus-free on peach seedlings until the spring of 1961. Of the 'sound' seeds from the trees in this group, virus was detected in 3.5% (0-10%) (Table 1). These results indicate the proportion of 'sound' seeds with detectable virus that occurred on healthy trees in this orchard at a time when natural spread was almost maximum. Virus spread was so rapid that 9 of these 10 trees showed initial shock symptoms in the spring of 1962. These trees were probably infected during the 1961 blossom period (6). Although 100 'sound' seeds were tested from one of these 9 trees, no virus was detected and yet the tree developed shock in 1962. On the other hand, 4% of the seeds from another tree that remained virus-free, carried virus. As this tree first showed shock in 1964, it probably was infected during the 1963 blossom period. Therefore, though the number of diseased seeds on a healthy tree may indicate the degree of cross-pollination with virus-carrying pollen, it is not a reliable indicator of the tendency for a tree to become infected. It must be remembered that the samples tested were very small and that trees with only a very few fruits can become infected (6, 8). In 1961, yield in the test orchard averaged 100 lb (45,359 g) of fruit per tree. The mean weight of 200 fruits was 755 g. Based on the study of variation in Montmorency cherry fruit size, conducted by Davidson and George (2), the average number of fruits per tree was estimated at just over 12,000. Therefore, the 50 seeds assayed from all trees but one, represent only 0.42 % of the estimated total fruits on an average tree. George and Davidson ( 6) obtained infection in a tree th"' monmd only thm frui1'. More mcntly Gilm0< (8) ropmted infrction

so+ C\:-IADIAK JOC:RNAL OF PLANT SCIENCE [Vol. 46 of two trees that matured only 8 and 11 fruits respectively. Both reported that trees maturing many more fruits failed to become infected. Thus failure to detect virus in the small, 100-seed sample of the one tree as reported above does not mean that it did not carry diseased seeds in sufficient number to cause infection. In fact, it appears that infection is not dependent upon a certain number of infected seeds but rather upon some obscure mechanism or set of conditions that allows the virus to pass from the pollen into the mother tree. Probably only one infection site is sufficient. It may be, as suggested by George and Davidson ( 6), that seed infection is not a prerequisite for tree infection. Aborted Seeds The average number of aborted seeds was lowest on the healthy trees (Table 1). Those trees with shock in 1961 had almost twice as many aborted seeds as the healthy trees. About one-third of the seeds from trees with SCY symptoms were aborted. Those trees with NRS symptoms produced fruits with fewer aborted seeds than those on trees with SCY but more than those on healthy trees. Thus, virus infection, especially SCYV, tended to increase the abortio n rate of Montmorency cherry seeds. Recently Attafuah ( 1) has shown a very high degree of seed abortion in fruits from sweet cherry trees with tatterleaf caused by a NRSV. As he suggests, there may be a considerable difference between the sensitivity of sweet and sour cherries to embryo abortion as induced by virus. The diffe ; ence between the results of Attafu h and those reported in this paper would support this theory. ACI NOWLEDGMENTS Jn this, the final in a series of papers on necrotic ring spot and sour cherry yellows, we wish to thank the many growers who allowed us to use their orchards for detailed studies on the spread and symptomatology of virus diseases. In particular we wish to thank Mr. E. D. Troup whose trees, in addition, were used in the seed tests reported here as well as in deblooming experiments reported previously. This research could not have been done without the cooperation of growers. We also thank M. G. Howard, Technician, Research Station, Vineland Station, Ont., for help throughout this study. REFERENCES 1. A TTAFt:AH, A. 1965. Embryo abortion in virus-infected sweet cherry. F.A.O. Plant Protection Bull. 13, 79-82. 2. DAVIDSON, T. R. and GEORGE, J. A. 1959. Variation in weight, volume and diameter of sour cherry fruits from individual trees. Can. J. Plant Sci. 39, 210-214. 3. DAVIDSON, T. R. and GEORGE, J. A. 1959. Symptoms of sour cherry yellows and necrotic ring spot in relation to time of inoculation. Can. J. Plant Sci. 39, 431-436. 4. DAVIDSOK, T. R. and GEORGE, J. A. 1964. Spread of necrotic ring spot and sour cherry yellows viruses in Niagara Peninsula orchards. Can. J. Plant Sci. 44, 471-484. 5. GEORGE, J. A. 1962. A technique for detecting virus-infected Montmorency cherry seeds. Can. J. Plant Sci. 42, 198-203. 6. GEORGE, J. A. and DAVIDSON, T. R. 1963. Pollen transmission of necrotic ring spot and Will chrny ydl= from mo-rn-mo. Cm. ]. Pbm Sd. "-' 276-288.

September 1966] GEORGE AND DAVIOSON-VlRl.TS ASSAY OF SEEDS 505 7. GEORGE, J. A. and DAvmsoN, T. R. 1964. Further evidence of pollen transmission of necrotic ring spot and sour cherry yellows viruses in sour cherry. Can. J. Plant Sci. 44, 383-384. 8. GILMER, R. M. 1965. Additional evidence of tree-to-tree transmission of sour cherry yellows virus by pollen. Phytopathology, 55, 482-483. 9. GILMER, R. M. and WAY, R. D. 1960. Pollen transmission of necrotic ringspot and prune dwarf viruses in sour cherry. Phytopathology, 50, 624-625. 10. GILMER, R. M. and WAY, R. D. 1961. Pollen transmission of necrotic ringspot and prune dwarf viruses in cherry. Tidsskr. for Planteavl, 65, 111-117. 11. GILMER, R. M. and WAY, R. D. 1963. Evidence for tree-to-tree transmission of sour cherry yellows virus by pollen. Plant Disease Reptr. 47, 1051-1053.