Transmission of Bean common mosaic Virus by Seed of Phaseolus vulgaris L. cultivar Beka

1. In this study an attempt was made to elucidate why transmission of bean common mosaic virus (BCMV) by seed of Phaseolus vulgaris L. cultivar Beka is always restricted to a certain percentage of seeds harvested from diseased plants. 2. The development of the healthy ‘Beka’ plant, especially the formation of flower buds, pods and seeds, was analyzed. Special attention was given to the development of the ovaries and ovules from their primordial stage until some days after fertilization. 3. The influence of infection upon the development of shoots, flower buds and seeds was studied. Inoculation of the plants before the 1st compound leaf at the main stem had fully developed resulted in initial retardation of growth followed by a vigorous development of sprouts. All compound leaves showed mosaic symptoms. Pods formed on these plants often were malformed by non-development of ovules and seeds. Plants which became infected in later stages showed necrosis of flower buds and of definite parts of the main stem, shoots and leaves. The developmental stage of the plants determined the sites at which necrosis would occur. Plants which became infected after all compound leaves at the main stem were developed never showed symptoms. The relation between the developmental stages of leaves, internodes and flower buds at the time they became infected, and the character of the symptoms they later developed was studied in detail. 4. It became apparent that the first two compound leaves of the main stem each could show the following symptoms: a. a coarse mosaic with curling if it became infected during an early stage of development, during which mainly cell-divisions occur. b. a fine mosaic with only slight deformations if a leaf became infected somewhat later, but still in an early stage of development. c. chlorotic and necrotic symptoms if a leaf became infected during the grand period of growth, in which mainly cell-elongations occur. d. epinasty without any discoloration or malformation if a leaf became infected at the end of the grand period of growth. e. no symptoms if a leaf had matured or nearly matured before infection took place; virus was, however, present in the leaf. 5. Necrosis of shoots and main stem turned out to be related to the grand period of growth of internodes at the time virus reached an infectious level in the plant. Basal internodes are the first to pass through this period of strong enlargement, successively followed by internodes situated more acropetally. Therefore, the older the plants were at the time of infection, the more the necrotizing parts of the main stem and shoots moved in an apical direction, until at last all internodes had passed the critical period and acro-necrosis occurred no more. 6. The relation between the stages of development at the time of infection and the character of the subsequent symptoms is far more difficult to study with flower buds, which contain so many heterogenous organs, than with stems, shoots and leaves. Flower buds became necrotic when infection occurred during the period at which the embryo-sac within the ovules developed from a mono-nucleate into an eight-nucleate state. It could not be determined whether this necrosis and dropping of buds must be considered as a result of virus infection of the strongly enlarging peduncles being in the grand period of growth or of the simultaneously enlarging ovaries, ovules and embryo-sac. However, it became clear that buds with ovules between the mono- and eight-nucleate stage when invaded by virus, could be disregarded as to the production of infected seeds. 7. All those flower buds which had passed the critical period of necrosis] at the time they became infected produced healthy seeds. Infected seeds were produced only by those flower buds which had not yet reached the critical period of necrosis at the time virus reached an infectious level in the plant. Therefore to obtain infected seeds, inoculation had to be performed in an early stage of development. The infection of, and transmission by seeds of even these buds, however, averaged only 15 %. Factors which might be responsible for this low percentage were studied. 8. Although virus turned out to be inactivated in pod-walls and seed-coats during maturation and drying, it did not seem to be inactivated in the embryos during maturation and drying, nor during storage and germination of the seed. Therefore, the low percentage of seed infection probably is not due to inactivation of the virus. 9. Cross-pollination experiments revealed that embryo infection might originate from an infected egg-cell or an infected pollen grain. Because the percentage of infected seeds did not appear to change during maturation and drying of the seed, it had to be supposed that the low percentage of seed infection is due to the fact that only a low percentage of mega- and microspores becomes invaded with virus. The percentage of infected seeds which originated from artificial cross-pollinations between a healthy and a diseased plant turned out to be twice as high as the percentage of infected seeds which originated from self-pollination. 10. A study was made of why so low a percentage of the egg-cells of plants which were infected in an early stage of development, became invaded with virus. No virus particles were detected in dip-preparations of ovaries which had not yet differentiated into ovary-walls and ovule-primordia. Their still-meristematic character may be related to the absence of infectious virus. Virus particles could be detected in dip-preparations of about 80 % of the ovaries in which ovules just started to develop, prepared 20 days after inoculation of the plant. So the low percentage of infected egg-cells cannot be due to the fact that a large proportion of the ovaries does not become infected. Virus particles could be observed in dip-preparations of about 80 % of the ovules 2 to 3 days before fertilization. In these ovules, and also in a high percentage of young seeds, virus reached an infectious level. As infection of the embryos averaged about 15 %, however, a barrier was supposed to be present in nearly mature ovules some days before flowering and after fertilization, preventing the entrance of virus into the egg-cell and later into the embryo. It may be that only a low percentage of young ovules becomes infected before this barrier is formed. This might explain the low percentage of egg-cell infection. The character of this barrier was not studied with these buds but with those which never produced infected seeds, described under 12, 13 and 14. 11. The cause of the reduction in seed production of plants infected in an early stage of development might also be partly responsible for the low percentage of seed infection. Therefore this phenomenon was examined. The reduction of seed production appeared to be due to: a. a decrease in the number of ovule-primordia initiated per ovary. b. an inhibition of the development of young seeds. c. a non- or inadequate fertilization of ovules. As the phenomena mentioned under a. and b. could also be observed with healthy plants grown under deficient light, they may be due to a lack of nutrients. A high percentage of non- or inadequately fertilized ovules was observed only with plants infected with BCMV. In what way the presence of the virus may be responsible for this phenomenon is unknown. Anyhow, the phenomenon can only be of secondary importance in determining the percentage of infected seeds. 12. Experiments were carried out to examine why flower buds never produced infected seeds if they had passed the critical period of necrosis at the time they became infected with virus. The time of infection of these flower buds, their ovaries and ovules was determined in relation to the moment of fertilization. The anatomical development of the ovules was studied from the moment virus material entered the ovules until virus reached an infectious level within them. 13. Virus material entered the ovaries 7 or 8 days before flowering, at the time they contained ovules with a four-nucleate embryo-sac. Infectious virus could be demonstrated for the first time within these ovaries 4 or 5 days before flowering. At that time their ovules contained an eight-nucleate embryo-sac. Infectious virus could be demonstrated within the ovules themselves for the first time 2 or 3 days before flowering. At that time they contained an eight-nucleate embryo-sac in which the polar nuclei had already migrated to the middle. Because flower buds comparable to those of which the infection of ovaries and ovules was studied produced only healthy seeds, the hypothesis was proposed that a barrier might be within the ovules which might prevent infection of the egg-cell and later of the embryo. 14. Light-microscopical and electron-microscopical examination of the embryosac and the embryo-sac-surrounding tissues of ovules 2 to 3 days before flowering, revealed that the nucellar tissue had started to disintegrate already some days before fertilization, leaving the lateral sides of the embryo-sac in immediate contact with the inner integument. No plasmodesmal-like structures were observed in cell-walls of the inner integument bordering the embryo-sac. If virus material can be transported from cell to cell only by plasmodesmata, it probably cannot enter the embryo-sac through the inner integument. Entrance through the chalazal end of the embryo-sac may be difficult or impossible as at this site the nucellar tissue also has already started to disintegrate before fertilization. 15. The disintegrating micellar tissue, and the medium surrounding the egg-cell and later the embryo, may prevent virus multiplication and virus-transport. This fact, together with the impossibility of virus material to enter the embryo-sac through the integuments, may be considered as a barrier which prevents the infection of egg-cell and later the embryo. In this way it can be explained why flower buds which become infected just before or after fertilization never produce seeds infected with BCMV.