This a first draft, all comments, suggestions, questions, etc. welcome (unfortunately, my formatting, use of bold face, etc. was removed when I uploaded the manuscript).
Can North American rose hybridizers safely use pollen from PNRSV infected roses?
By Henry Kuska
First Draft, 2-08-2003
In the 1990s I ran a (free) e-mail rose breeding scientific literature “course”. One of the questions that I received was from a prominent North American rose hybridizer (of All American Rose caliber). He stated that from what he had read rose mosaic virus could not be spread by pollen. However, he had occasionally observed virused roses among his seedlings. He asked if I knew of any scientific rose literature on the subject.
First, I will define my terms. There are a number of viruses that historically have been included under the broad term “rose mosaic virus”. In this discussion, I will limit myself to what, at present, appears to be the most prevalent rose “mosaic” virus in North America, prunus necrotic ringspot virus (PNRSV). A more accurate scientific name is prunus necrotic ringspot ilarvirus, but I will use the more common prunus necrotic ringspot virus.
In the “Plant Viruses Online” database, the (general) modes of transmission for PNRSV are given as: “Transmitted by means not involving a vector. Virus transmitted by mechanical inoculation; transmitted by grafting; not transmitted by contact between plants; transmitted by seed (to over 80% in Prunus pennsylvanica but much less in peach); transmitted by pollen to the seed and transmitted by pollen to the pollinated plant.” (1)
The above is a general statement. It is possible (but not probable) that PNRSV could be transmitted in pollen of other infected plant species (to some non-zero degree) but not transmitted at all (zero) in roses. To see whether this remote possibility of zero transfer is actually the case, specific scientific studies of roses and PNRSV will be examined.
The earliest pertinent studies that I could find were three that were published in 1980.
One was by D. J. Barbara, East Malling Research Station, United Kingdom. She reported that “In rose high levels of virus were present in petals and stamens but only very low levels in the sepals.” Note, for the non scientific reader, pollen is released from the stamens. Also, she is referring to PNRSV. (2)
The second 1980 paper is actually 3 papers published by J. B Sweet, Long Ashton Research Station, University of Bristol,United Kingdom. He reported that PNRSV was detected in the pollen of Peace and Queen Elizabeth roses, and in 1 % of two batches of seedling Rosa multiflora rootstocks. As is typical of scientific caution (or at least should be), he points out that finding the virus in the seedlings is not a definitive proof of transmission through the rose seed. However, his statement should not be interpreted that he did not find non grafted seedlings with virus, it means that he cannot be sure that it came through the seeds; it could of come from infected shears, thrips, etc. after the seedlings were planted. What it does show is that there was transmission of PNRSV to non grafted seedlings in 2 different batches (unless there was experimental error). Since multiflora seedlings are grown together in a field, I think that one cannot use “root grafts to an infected neighbor plant” as a probable reason as you would be going in a circle logically i.e. you would have to explain how the infected seedling that supplied the root graft got infected. In the Journal of Horticultural Science paper, he states: "“Two plants from a batch of about 200 Rosa multiflora seedling rootstocks with mosaic symptoms on their leaves induced PNRSV-type symptoms in the woody indicatore, whereas four symptomless plants did not. Virus was sap-transmitted from one of the mosaic-diseased R. multiflora seedlings which induced typical PNRSV symptoms in herbaceous test plants, and infected cucumber sap-precipated with NRSV-G but not with ApMV-P and PDV-B antisera.”
Note, there is no graft on these infected plants; they are seedlings. Also note, Sweet used a very crude detection systems (visual symptoms), we do not know how many more of the 194 were infected but did not show symptons. Even of the 4 symptomless plants that he tested and that were negative, we do not know if any of them would have tested positive with todays’ much more sensitive procedures. This paper is a very important one relative to the topic being considered! (3)
The third 1980 paper was published by B. J. Thomas. He found that both the gel immunodiffusion test and the latex test were unable to detect PNRSV in infected roses but that both ELISA (enzyme-linked immunosorbent assay) and SSEM (serologically specific electron microscopy) methods were able to (in some cases ELSIA also failed). SSEM was twice as sensitive as ELISA. Of particular importance here is that he was able to detect the virus in the the rose stamens. (4)
The next two papers are also very important to this discussion as the first reports (and the second confirms) that it is possible to “transfer the virus by mechanical means”. This is an important finding because this removes the possibility that the virus is “too fragile” to be transferred by anything but a graft (living phloem cells of the plant). The “too fragile” theory seems to be a key assumption of the “no transmission but by grafting” school of thought.
The first report of mechanical transfer was in 1981, by B. J. Thomas. He reported that his preliminary attempts to transfer the virus by mechanical means failed, but by adjusting his experimental procedure he was successful - including the use of infected rose petals and anthers (it is not clear if the petals and anthers were mixed together or tested separately). (5)
The ability to transfer the virus by mechanical means was confirmed in 1994. H. Baumgartnerova, Institute of Experimental Phytopathology and Entomology, Slovak Academy of Sciences, Czechoslovakia reported that the virus was positively mechanically transmitted from diseased leaves and pollen of roses. (6)
In fairness to possible overlooked articles, I should point out that my search may not be complete as computer abstracting searches are relatively recent; and there may be articles in relatively small scientific journals that have not yet been covered by the abstracting services nor referenced in the articles that I have cited. However, I feel that the above six set of articles provide sufficient information such that North American rose hybridizers should be wary of using PNRSV diseased roses as pollen parents in their hybridizing program.
Examples of how others have interpreted the literature up to the date of publication of their own work are given next.
In the 1983 book “Compendium of Rose Diseases” by R. K. Horst, Professor Horst (Cornell University, Plant Pathology) wrote: “PNRSV is pollen-transmitted in fruit trees. Pollen transmission is suspected to occur in roses also. Since spread in the field is slow.” (7)
Baldo Villegas (Associate Environmental Research Scientist (Entomologist),California Department of Food and Agriculture) consulted with the following plant pathologists with the California Department of Food and Agriculture’s Plant Pests Diagnostic Centre: Dan Opgenorth and Dennis Mayhew before preparing his web page article on the subject. He stated: " They have given me invaluable advice in preparing this article." Regarding spread, he stated: “Some pathologists suspect that mosaic may be pollen transmitted which could prompt removal if other roses in the garden are valuable and not already infected”. (8)
In 1989 in an article titled “Incidence of Rose Viruses in Spain” M. Cambra, J.L. Martinez-Torres, M.J. Benaches, E. Camarasa, and M.T. Gorris studied 4,730 rose samples. They found 4.2% of the roses had PNRSV. The breakdown was: 44.0 % of the minatures, 1.1 % of the hybrid teas, and 1.5 % of those budded on Manetti rootstocks. Cambra et.al. state: “The high rate of PNRSV contamination in minature varieties seems to be associated to their long existence.” Later in another paragraph they say:“…since this virus is pollen transmitted (in addition to grafting).” They also suggest that one way to prevent the virus from spreading is to prevent the plants from flowering. (9)
Professor Gerald C. Adams (Department of Plant Biology and Department of Plant Pathology, Michigan State University) makes the following statements: “Rose Mosaic Virus in the Americas is most commonly caused by prunus necrotic ringspot virus which is a pollen transmitted virus but transmission by pollen is very low. When transmission by pollen does occur it is usually due to a high population of thrips. The thrips carry the pollen or infected sap and introduce it into a feeding scar on a leaf or petal.” Farther in the article he states: “Spread of PNRSV by pollen is measurable in cherry orchards and the virus has been found to exist at high titer in rose stamens. Never the less, field spread in rose nurseries has not been easily demonstrated and apparently is rare. In fact most spread of PNRSV is by grafting during vegetative propagation.” (10) I included the second quote for completeness, but please remember that the focus of the present paper is spread in hybridizing, not field spread.
Unfortunately, nature often is not as simple to understand as we would like. This appears to be one of those times as there are 2 papers that looked for PNRSV transfer to seedlings through pollen but did not find it.
The first paper is Sweet’s Journal of Horticultural Science paper referred to earlier. He germinated seeds from the two infected plants (36 seedlings from one and 24 seedlings from the other. None showed visual symptons and 5 random samples from each were grafted on P. Persica GF 305 seedlings (virus test plants) and did not test positive. There are several reasons that I do not consider this report definitive: 1) 10 grafting tests is too small a sample size, 2) the use of visual symptons on 36 samples can now be considered as both too small a sample and to be deficient in test sensitivity, and 3) if the infected bushes were among the 198 "healthy ones; it is possible that the pollen came from the healthy bushes since many (most, all?) pure species roses are self sterile (see discussion 2 paragraphs down).
The second virus-seed transmission test paper was published in 1984, by B. J. Thomas. (11) He reports on three different experiments that could provide some information regarding virus-seed transmission. In the first experiment he crushed 10 seeds from each laboratory infected virused plant with hips, and could not, using ISEM, detect any PNRSV in the seeds (he did not state the number of batches examined). In the second experiment he examined, with ISEM, 1067 seedlings grown from seeds harvested from the infected bushes and found no PNRSV infected plants. A possible explanation for the failure to detect PNRSV in the seeds or resulting seedlings is that the seeds may have also (like Sweet’s samples) been unintentionally produced from non-infected pollen. The following is Thomas’ description of the plantings: “Seed was collected for 3 yr from virus infected plants planted in nematode-tree soil 50 cm apart in two rows 2 m apart so that plants of the same species were opposite each other. A row of corresponding healthy roses were planted equidistant between these rows.” The roses utilized were all species roses, R. canina, R. canina, var. Brogs, R. corymbifera, R. multiflora, and R. Rugosa. If we accept the self sterile model, the seeds that he should of been examining were the ones on the non virused bushes between the two infected ones. (His experiment may still be valid if the neignboring plants in the same “infected” row were of the same species and also had PNRSV - I have to add the “also had PNRSV” because he had plants with 3 different virsus in the study - the neighbors could have had one of the other 2 viruses. This information was not given. Also, except for R. rugosa, the other species roses are once bloomers, and the bloom periods for the same row infected neighbors may possibly not have overlaped the bloom period of the one between in that row. In the third “experiment” he did detect PNRSV in three seedlings of new rose cultivars (planted in the Royal National Rose Society’s trial grounds). One infected plant was of unknown origin, one was from France, and one was from the USA (via France). Since the hybridizing, growing conditions, were not controlled (for example, was a virused rootstock used?), the results from this third “experiment” are not scientifically of value relative to the present question.
This paragraph discusses the literature evidence for the self sterile model. In 1986 P. Cole and B. Melton published a paper which investigated the ability of rose pollen to fertilize flowers on the same bush. (12) The diploid species were all highly self steile. None of the 23 diploid specimens exhibited over 4 % fertility and 18 of the 23 produced no self-set seed. They also studied the fertility with pollen from another plant of the same species and found that the diploid group was 50 times more cross compatable than self compatable. For roses of higher polyploid level 12 of the 16 studied were no more self compatible than the diploid group. Unfortunately, only two of the species studied by Thomas was also studied by Cole and Melton, one was R. rugosa which was 100 % self sterile, and the other was R. rubiginosa Linnaeus (R. eglanteria) which was 83.4 % self sterile (one sample). Thomas’ R. canina, R. canina var. Brogs, and R. corymbifera are of the canina section as are two of the species studied by Cole and Melton. One is R. horrida Fisch. which was found to be 100 % sterile and the second is R. laxa Froeb. which was one of the least self sterile species studied, only 55 % self sterile. It should be noted that only one plant of R. laxa Froeb. was available for this study. In the other case of low self sterility R. engelmanni, one sample was 54 % self sterile while four other samples of the same species were 100 %, 99 %, 100 % and 100 % self sterile. Thus, the R. laxa Froeb. low self sterility number may not be representative of the species, but actually due to having an individual plant that has some other species in its ancestory.
A very recent paper by Han YounYol and Yu SunNam (13) did confirm that R. Rugosa was self sterile but reported that R. multiflora did self fertilize. I posted a request for members of the Rose Hybridizing Association to check to see if they had information on the question of whether R. multiflora is self sterile. David Zlesak stated that" roses have a gametophytic self incompatibility system which can be easier to break down than a sporophytic system…" John Moe replied that he had an isolated R. multiflora that did not have any hips all season. Jim Turner reported a few hips, but his bush was not isolated (he reported an early spring blooming, one textbook source reports a July blooming?). Paul Barden reported 2 separated bushes had many hips. The bushes were not isolated from other roses. He had grown seedlings from the hips and the seedlings looked like they were only R. multiflora. Possible complications that may explain Paul Barden’s results include: 1) apparently first generation crosses with species roses often look like the species rose. It is not until the second generation that major differences appear, 2) it is possible that a small amount of compatible pollen (from a neighboring bush) may break the barrier for pollen that would normally be incompatable, and 3) Paul’s R. multifloras may already have some foreign blood in them as over the years rootstocks have been selected for various characteristics such as lack of thorns.
There is a very recent paper which I am including for completeness, but it does not specifically address the pollen issue. It is a 2001 paper by Benoit Moury, Loic Cardin, Jean-Paul Onesto, Thierry Candresse, and Alain Poupet. They report that 4 percent of cut-flower roses from different European sources were infected with Prunus necrotic ringspot virus (PNRSV). The abstract reads: “Progression of the disease under greenhouse conditions was very slow”. Now, the abstract is all that most scientists will read so most (all?) authors proofread it very carefully - in other words I would not expect an error in a abstract. However, in the article the following appears: “Our observations show that the progress of PNRSV infections in roses grown in greenhouses is very slow (or nonexistent), because in our trial only 1 % of the plants were PNRSV infected 2 years after planting (without taking particular cautions for isolation of the plants or disinfection of the tools used for pruning).” (14) What is confusing to me is the addition of the “(or nonexistent)” which does not appear in the abstract.
A possible reason that the spread is very slow through seed transmission is that the PNRSV appears to be on the surface of the pollen and in the part of the pollen that forms the pollen tube but not in the actual “sperm”. (15)
A definitive experiment is to hand pollinate a fertile isolated rose (that has had its own pollen removed) with diseased pollen from an infected rose that is known to cross with it. The pollinated flower would then be covered to prevent stray pollen from contaminating the experiment. This would check for seed transmission.
Reference (1) http://image.fs.uidaho.edu/vide/descr658.htm , Brunt, A.A., Crabtree, K., Dallwitz, M.J., Gibbs, A.J., Watson, L. and Zurcher, E.J. (eds.) (1996 onwards). `Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20th August 1996.’ URL http://biology.anu.edu.au/Groups/MES/vide/ Dallwitz (1980) and Dallwitz, Paine and Zurcher (1993) should also be cited.
Reference (2) Barbara, D., J., Acta Phytopathologica Academiae Scientiarum Hungaricae, volumn 15, pages 329-332, (1980). Reprinted in Acta Horticulturae, volumn 94, published March 1, 1981.
Reference (3) Sweet, J., B., Acta Phytopathologica Academiae Scientiarum Hungaricae, volumn 15, pages 231-238, (1980). Reprinted in Acta Horticulturae, volumn 94, pages 231-238, (1981).
He published a similar (but not just a duplicate) paper in: Journal of Horticultural Science, volumn 55, pages 103-111, (1980).
Reference (4) Thomas, B. J., Annals of Applied Biology, volumn 94, pages 91-102, (1980).
Reference (5) Thomas, B.J., Annals of Applied Biology. volumn 98, pages 419-429 (1981).
Reference (6) Baumgartnerova, H., Acta Horticulture, volumn 377, pages 357-359, (1994). ROSA SP. - RESERVOIR OF THE SOUR CHERRY NECROTIC RINGSPOT VIRUS
Reference (7) Horst, R., K., book “Compendium of Rose Diseases”, published by The American Phytopathological Society, St. Paul, Minnesota. pages. 26-27, (1983) .
Reference (8) Baldo Villegas web page, http://members.tripod.com/buggyrose/ipm/83rosemosaic.html
Reference (9) Cambra, M., Martinez-Torres, J.L., Benaches, M.J., Camarasa,E., Gorris, M.T., Acta Horticulturae, vol 246, pages 309-312, (1989).
Reference (10) Adams, G., C, “Rose Mosaic Virus in the Nursery”, http://extension.bpp.msu.edu/rosemosaic/ , last revised 9/7/2002.
Reference (11) Thomas, B., J., Annals of Applied Biology. volumn 105, pages 213-222 (1984).
Reference (12) Cole, P., Melton, B., J. Amer. Soc. Hort. Sci., volumn 111, pages 122-125, (1986).
Reference (13) YounYol, H. and SunNam, Y., Journal of the Korean Society for Horticultural Science, Volumn 43, pages 326-332, (2002).
Reference (14) Moury, B., Cardin, L., Onesto,Jean-Paul, Candresse, T., Poupet, A., Phytopathology, Volumn 91, pages 84-91,(2001)
Reference (15) Aparicio, F., et.at., European Journal of Plant Pathology, volumn 105, pages 623-627, (1999).