Lack of seed transmission of Cymbidium Mosaic Virus in Dendrobium

Lindleyana 11(4): 211- 213, 1996

Reprinted by permission from the author

LACK OF SEED TRANSMISSION OF CYMBIDIUM MOSAIC VIRUS IN DENDROBIUM

KATRINA G. PORTER (2), ADELHEID R. KUEHNLE (2,3), AND JOHN S. HU (4)

2 Department of Horticulture, University of Hawaii, 3190 Maile Way, Honolulu, Hawaii 96822 U.S.A.
3 To whom requests for reprints should be addressed.
4 Department of Plant Pathology, University of Hawaii, 3190 Maile Way, Honolulu, Hawaii 96822 U.S.A.

ABSTRACT: Dendrobium hybrid seedlings derived from parent plants infected with Cymbidium mosaic potexvirus (CyMV) were tested for the presence of CyMV to determine if seed transmission occurred during green capsule culture. ELISA tests on 7,050 seedlings of seed-propagated D. Jaquelyn Thomas 'Uniwai Supreme' and 'Uniwai Prince' were negative for the virus, indicating that CyMV is not seed-transmitted.

CYMBIDIUM MOSAIC POTEXVIRUS (CyMV) commonly infects many members of the Orchidaceae. When expressed, foliar symptoms of CyMV include chlorotic mosaic and necrosis and floral symptoms include necrotic flecks, color break, and flower distortion (Lawson, 1995: Okemura, Kamemoto and Ishii, 1983; Wisler, 1989; Yuen, Kamemoto and Ishii, 1979). CyMV can reduce plant vigor and cause irregular blooming (Wisler, 1989).

CyMV is a stable, highly infectious virus that is mechanically transmitted. CyMV systemically infects all parts of the orchid plant, including leaves, flowers, pollinia, roots, and epidermal cells (Kado and Jensen, 1964: Lawson and Hearon, 1974). Seed-transmission of the virus has not been documented (Wisler, 1989). However, it is thought that some seed may become infected during the common green pod or green capsule culture, in which scraping of infected maternal tissue during seed removal may transmit virus (Yuen et al., 1979).

Although the report by Yuen et al. (1979) suggested that one out of 60 mature plants from two 2 1/2 -month-old capsules was infected with CyMV, to date no comprehensive study utilizing large numbers of seedlings has been done. In addition, because of the low occurrence of infection and lack of precautions against virus transmission during greenhouse handling of the plants, the authors conclude that infection was likely due to cultural practices (Yuen et al., 1979; H. Kamemoto, personal communication).

The purpose of this study was to determine whether or not CyMV is transmitted via seed to seedlings in Dendrobium orchids.

MATERIAL AND METHODS
Seedlings of the commercially important University of Hawaii Dendrobium hybrids D. Jaquelyn Thomas 'Uniwai Supreme' (UH232) and D. Jaquelyn Thomas 'Uniwai Prince' (UH503) (Kamemoto, 1985) were used. The capsule and pollen parent plants of each cultivar were determined to be CyMV-positive by enzyme-linked immunosorbent assay (ELISA) tests.

To produce seedlings of UH232 and UH503, flowers were cross-pollinated and
2½ -month-old green capsules were harvested. Capsules were surface-sterilized by dipping in 95% ethanol and flaming briefly. Immature seeds were mechanically separated aseptically from the placenta ridge by scraping the inner capsule. These were germinated on Vacin and Went (1949) medium modified with 7.5 g/liter agar, 75 g/liter banana, and 15% coconut water (Sagawa and Kunisaki, 1984). Seedlings were subcultured twice to a final stage of 25 plants per 10-cm-square polycarbonate box (G&B Orchid Laboratory and Nursery, Vista, California). A total of 7,050 seedlings in 282 boxes were obtained: 2,875 seedlings of UH232 and 4,175 seedlings of UH503.

Table 1. Absorbance values at 405 nm in ELISA from Dendrobium seedlings assayed for Cymbidium mosaic virus.

2 hour							18 – 20 hour (overnight)
Plate	Seedlings	+3 s.d.^(b)	CyMV-positive controls		CyMV-negative control	Negative control X2	Seedlings	+3 s.d.	CyMV-positive control	CyMV-negative control	Negative control X2
1	0.021	0.085	0.967	(1:250)	0.009	0.018	0.085	0.330	***	0.185	0.370
2	0.006	0.013	0.467	(1:250)	0.022	0.044	0.019	0.038	1.560	0.074	0.148
3	0.000	0.006	0.693	(1:250)	0.009	0.018	-0.001	0.011	***	0.023	0.046
4	0.090	0.103	1.318	(1:250)	0.096	0.192	0.111	0.143	***	0.120	0.240
5	0.005	0.021	1.526	(1:250)	0.010	0.020	0.011	0.065	***	0.029	0.058
6	0.004	0.024	0.229	(1:10)	0.002	0.004	0.029	0.102	0.897	0.019	0.038
7	0.008	0.012	0.326	(1:500)	0.007	0.014	0.014	0.032	1.065	0.013	0.026
8	0.003	0.007	0.782	(1:500)	0.001	0.002	0.008	0.014	***	0.001	0.002
9	0.013	0.032	0.488	(1:20)	0.012	0.024	0.023	0.080	2.044	0.018	0.036
10	0.003	0.009	0.594	(1:20)	0.001	0.002	0.006	0.037	2.113	0.003	0.006
11	0.001	0.004	0.793	(1:20)	0.005	0.010	0.009	0.022	***	0.001	0.002
12	0.009	0.013	0.199	(1:1000)	0.009	0.018	0.012	0.033	0.805	0.013	0.026
13	0.001	0.006	0.153	(1:1000)	0.005	0.010	0.013	0.037	0.711	0.030	0.060
14	0.006	0.010	0.178	(1:1000)	0.005	0.010	0.019	0.048	0.759	0.017	0.034
15	0.000	0.002	0.098	(1:1000)	0.008	0.016	0.002	0.006	0.361	0.018	0.036
16	-0.002	0.007	0.135	(1:1000)	-0.005	-0.010	-0.002	0.021	0.549	-0.006	-0.012
17	-0.007	-0.001	0.196	(1:1000)	0.002	0.004	-0.023	-0.002	0.879	-0.004	-0.008

a 17 multiwell plates were used to sample 7,050 seedlings in 282 groups; data from these groups were pooled and averaged.
b +3 s.d. means the value in the previous column plus three standard deviations
c Value is that of the lowest dilution on the plate, indicated in parenthesis
d *** indicates that the A(dbs) value was too high to quantify

ELISA tests were conducted to determine the incidence of CyMV among the seedlings. Double antibody sandwich ELISA (DAS-ELISA) was performed according to Clark and Adams (1977) in 96-well Immulon 2 "U" plates (Dynatech Laboratories, Chantilly, Virginia). Rabbit anti-CyMV polyclonal antibodies conjugated with alkaline phosphatase were used for detection of the virus (Hu et al., 1993). Sensitivity tests with these antibodies indicated a routine dilution end point of the virus extract of 1:10,000. All dilutions of the positive controls were made with extract from the CyMV-negative orchid controls. Since seedling leaves were sampled in groups of 25, an additional test was performed to determine that virus in one 0.1 g leaf disc from an infected plant, when combined with 24 healthy discs, could be detected when ground in 20 parts extraction buffer for a final dilution of 1:500 per disc.

Seedlings ranging in height from 2.5 cm to 12.5 cm were sampled once over a period of six months to two years after germination. Leaf samples were pooled into groups of 25 (each group corresponding to the 25 plants per box), weighed, and ground at 1:20 dilution in PBS-Tween for a final dilution of 1:500 per leaf. Two replicate wells were loaded per sample. A total of 17 multiwell plates were used to sample all 282 groups. Absorbance values at 405 nm were obtained after 2 hr and after 18 to 20 hours (overnight). Extract of known CyMV-infected positive and CyMV-free negative controls, obtained from protocorm-like body tissue culture of D. Jaquelyn Thomas 'Uniwai Mist' (UH800) (Kamemoto, Amore and Sugii, 1990) and an unnamed hybrid 'M61', respectively, were diluted to at least 1:10 and were included on each plate. Sample values were considered negative if the average of the two replicate wells was less than 0.05 or was less than 2 times the average of the negative (healthy) control for both the two hour and overnight readings (Sutula et al., 1986) If the groups of 25 leaves had a positive reading, the plants were to have been tested individually. However, since all the group samples tested negative, individual plants were not tested.

RESULTS
All 282 groups representing leaf samples from 7,050 seedlings tested negative for CyMV by ELISA. For ease of presentation, data from all seedling groups on each plate were averaged and three standard deviations provided (Table 1). In all cases, the average value of the samples plus three standard deviations was not significantly different from the negative control (P < 0.05). All of the seedlings from cultivars 'Uniwai Supreme' and 'Uniwai Prince' involving CyMV-infected parents were CyMV-free as indicated by ELISA.

DISCUSSION
Virus-free seedlings of UH232 and UH503 were produced from flowers of CyMV-infected plants pollinated with CyMV-infected male parents. CyMV was not passed to the progeny through the seed capsule or pollen and is apparently not seed transmitted. These findings support those of H.F. Oakeley (Blowers, 1992) that seeds of the genus Lycaste are virus-free, whereas the pollinia, column, and other flower parts may contain virus. Moreover, the findings also support the conclusion by Yuen et al. (1979) that CyMV is not seed-transmitted in Dendrobium.

Dendrobiums are economically the most important orchids grown in the Pacific Basin region. Field surveys conducted in Hawaii showed that 4% of University of Hawaii seed-propagated plants less than three years old were infected with CyMV, where CyMV was detected in as high as 94% of the samples collected from 4 - 7 year old orchid plants (Hu et al., 1993). Since this paper has shown that orchid seedlings less than two years old are free of virus while in vitro, the 4% CyMV infection of plants three years old being field grown is believed to have occurred due to the mechanical transmission during potting and handling (Hu et al., 1994). Because harvesting of flowers starts when Dendrobium plants are 3 - 4 years old, it is assumed that CyMV mainly infects the orchids during harvesting (Hu et al., 1993). In contrast with areas where commercial cultivars are vegetatively propagated, the majority of Hawaiian cultivars are seed-propagated. Assurance of virus-free starting material for farmers is the first step in controlling viral disease. As there is no method currently available for orchid virus eradication during clonal propagation, and indexing is not routine, use of seed-propagated cultivars provides a most suitable mechanism for establishing virus-free plantings of orchids.

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