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The following recently appeared in a November 14, 2014 University of Florida Extension article titled:

Re-emergence of Rose Mosaic Disease in Florida Nurseries and Landscapes

The article includes the following statement:

"1.Always, start with clean stock plants and ideally, virus-indexed stock. However, be aware that RMD has been reported from healthy virus-indexed material in the past. The reason of why RMD is found in certified virus-free planting stock is not yet known."

http://nwdistrict.ifas.ufl.edu/phag/2014/11/14/re-emergence-of-rose-mosaic-disease-in-florida-nurseries-and-landscapes/

The above should be sufficient regarding the title of this paper; however, if interested, the following is a summary of what I had collected prior to the November 14, 2014 article.

The following was stated on a rose forum: "Purchasing virus indexed (VID) stock GREATLY enhances the likelihood that you will get disease-free roses. It is not, however, an absolute guarantee. I'm not the only one to have purchased a VID selection of a certain hard-to-find rose, only to see those unmistakable signs of virus appear as the rose matured."

From following thread:

http://forums2.gardenweb.com/forums/load/roses/msg022235431402.html

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This supports the following 1999 statement by PetRose:

"Nobody can guarantee that they sell "virus-free" roses since there is no test that can provide 100% assurance that a rose is virus free. I have heard Syl Arena speak on the subject of virus in roses and have spoken to him on the subject - his anti-virus commitment is one of the strongest in the industry."

Has PetRose changed his mind since 1999? The the following quote is from an article that was published in The Novus Newsletter of the Tidewater Rose Society (August 2010): "It is also important to understand that there is no test that can prove conclusively that a rose is not virused. As a consequence, the "guarantees" of virus-free roses offered by some nurseries that sell roses on their own roots are "hogwash." Certainly such nurseries can in-crease the odds that their roses do not have virus by careful selection of their stock and propagation of that stock on its own roots, but the claim that such roses are always without virus lacks candor."

AND

"An "indexed" block is a group of plants have been tested and have a very high likelihood of not having a virus."

Please notice that "very high likelihood" is not the same as 100%.

(In case you are a younger member and are not familiar with PetRose, he is "Robert B. Martin Jr." (University of Southern California School of Law (J.D. 1969)) who ran for American Rose Society Vice President and lost in a very close race to Jolene Adams. He was/is very active in the American Rose Society in Rose Exhibiting. )

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Are there other statements that support the above?

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Tom Liggitt stated: "Well, Malcolm, then you had better speak with the folks at Davis! Because some years ago they reported to the group at the innaugural meeting of a California rose plant grower's association meeing at Davis that virus DID in fact spontaneously reappear. AND said plants were in an screened, isolated green house, to boot!

I know what I heard that day at Davis, and other people in that audience

heard it, too.

https://groups.google.com/forum/?hl=en#!msg/rec.gardens.roses/VhgwCRP1rO0/Wb9k-OoUpRUJ

If you are not familiar with Tom Liggett, he was one of the founders of the Heritage Rose Garden.

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Is there an independent statement that supports what happened at Davis?

The following link contains the following 2005 statement by Dr, (medical doctor) James Sproul, the current General Director of the Rose Hybridizers Association: " I've had the opportunity to attend a couple of the GRC meetings, including one where various RMV's were discussed. I think that the reference to "supposedly clean blocks" was made in a discussion of varieties that had been heat treated. They were surprised to see virus infected plants showing up again after they had been indexed at Davis."

http://www.rosehybridizers.org/forum/message.php?topid=5948#6012

Dr. Sproul's hybridizing information is at: http://sproulroses.blogspot.com/

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The Foundation Plant Services, Davis, also have a 2005 document about rose virus testing. http://fpms.ucdavis.edu/WebSitePDFs/CustomServiceInfo&Agreements/RoseCustomServicesDescr&Prices.pdf . The following is a quote from it.

"Important Note:

Although the tests and procedures performed at FPS are state-of-the-art and executed with the utmost of care, they can detect only the specific viruses or other pathogens that are being tested for, and heat therapy cannot guarantee the elimination of all viruses or other pathogens that may be present in candidate materials. There may be other unidentified viruses, strains of already-known viruses, or other pathogens which can affect plant health for which tests are not performed or are not currently available. Some pathogens may replicate and move slowly in a plant, and may not be uniformly distributed throughout the plant at the time of sample collection. Therefore, disease tests should not be used as the sole factor in assessing plant health. Occasionally, disease may not be detected in only one growing season, so nurseries and growers are cautioned to inspect their plants carefully prior to propagation."

H.Kuska comment: "Occasionally, disease may not be detected in only one growing season, so nurseries and growers are cautioned to inspect their plants carefully prior to propagation." I interpret this as saying that: roses that we tested as virus free and shipped to you may occasionally actually show an infection after you received them - just another way of saying what the statements above indicate. The following is a more detailed description of the problem with missing low levels of virus concentration in plants in general:

Although the following quote from "Optimizing detection and management of virus diseases of plants" by Deborah M. Mathews, Ph.D., Author's affiliation: Assistant Cooperative Extension Specialist/Plant Pathologist, Department of Plant Pathology and Microbiology

University of California, Riverside, Riverside, CA 92521does not specifically mention roses, it sounds familiar to what has been reported for roses:

"These procedures work well to produce virus free materials most of the time, but my research has shown that virus levels may be temporarily reduced below the level of detection resulting in a negative virus test, but after some time (several weeks), as the plant matures, virus levels return to normal allowing a positive test result. This time frame means that initial screening will declare a plant as virus free, it is sold to growers and just about the time plants are ready for propagation they test positive and responsible growers eliminate them from the production cycle. This can cause huge losses in time and money especially if large scale propagation of cuttings has already occurred. Another pitfall is the presence of new, unknown viruses for which there are no available detection methods. If there are no symptoms and no appropriate tests, virus infections can be missed. Tissue culture however is the only method currently available to “rescue” desirable varieties from viral infections. Growers should buy from sources that utilize the best virus elimination and testing protocols and advertise that their products are virus tested. This doesn’t always guarantee that completely virus free plants will result, but it will be the best method available for obtaining healthy stock plants."

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THUS, the original statements regarding the Davis program has been confirmed by an actual 2005 Davis written document and reports by very reputable rosarians and scientists. This should be all that is needed. The reader may chose to stop here or look at the next section(s) for "interesting material" about rose testing and certification.

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The following Jul 23, 12 reply from Austin Roses was posted: "I can confirm that our varieties have not gone through the virus indexing program at UC Davis or the heat treatment program at Florida Southern College.

The major problem is that the Rose Growing Industry does not have a clear understanding of how the Rose Mosaic virus is spread. A lot of research has been carried out in this field but no substantive conclusions have been reached.

Our decision not to use these two above treatments is that there is no proof that they eradicate Rose Mosaic Virus. We see many rose growing fields in the United States throughout the course of a year. It is true to say that we have seen evidence of Rose Mosaic Virus in all of these crops.

We find it most effective to rogue out any virused plants in the field to keep the virus to a minimum."

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The following quote from a post on Fri, Nov 5, 2010 in the following link: http://www.rosehybridizers.org/forum/message.php?topid=31712#31864

states what another large nursery has been reported to say:

""I have an acquaintance here in Southern California. She is of the mentality, "absence of light black and Resurrection White", no gray areas in anything. She was, for many years, a rabid exhibitor, to the point of having multiple florist freezers in her home to hold her blooms for shows. She had dozens of portable gazebos to move around her garden to protect the buds as they formed. She was determined to grow uninfected varieties only as symptoms would disqualify her entries. J&P had begun their VI program and reserved the VI plants for specific states as the laws in those states had changed, making it illegal to supply virused stock across their state lines. She badgered J&P for their VI stock, then had it tested. Surprise! The results were that the VI stock was infected with the specific viruses tested for. She loosed her wrath on J&P and the PhD who ran the VI program. They replaced the plants, which, in turn, tested positive. When she contacted J&P again, she was told the program had been dismantled and the woman PhD in charge, reassigned as they found, even when grown in "sealed greenhouses with no possibility of spread, the viruses spontaneously regenerated". ------------------."

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The following is from a 2011 reviewed published scientific paper titled: "Elimination of viruses from fruit woody species and long term observation of health status."

http://agris.fao.org/agris-search/search.do?recordID=CZ2014000222

"However, totally 38% of pome fruit and 67% of sweet cherry seemingly virus-free clones after the first round of testing were positive in retests for the same viruses as in the initial plants. In the case of this material, the treatment probably depresses the amount of viral particles temporarily below the threshold level of detection of used diagnostic methods. In the course of further cultivation, the virus recovered-up and re-accumulated to a level of detection."

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Apparently heat treatment, by itself, sometimes may only reduce the concentration of virus to a level lower than can be detected by ELISA. This became clear when a method called PCR was developed. (PCR is 100 to 1000 times more sensitive than ELISA.).

H. Kuska comment: It is my understanding (from private e-mail) that at least one major rose testing university program ( the same one that switched from heat treatment/bud grafting to heat treatment/meristem tissue culture) switched several years ago from ELISA to PCR testing.

Here are some actual literature examples where a difference between ELISA and PCR detection was reported:

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"For the purpose of detection PNRSV in rose, IC-RT-PCR(immunocapture-reverse transcriptease-polymerase chain reaction)was established based on optimized double-antibody sandwich(DAS)- ELISA and RT-PCR to detect Prunus necrotic ringspot virus(PNRSV)from infected rose plants. Capture antiserum of PNRSV for virus immunocapture was conducted and a 760 bp fragment was amplified from diseased tissues with specific primers, designed on the basis of the coat protein gene of PNRSV.The fragment was cloned and sequenced. Sequence analysis result showed it shared 94 %～98% nucleotide identity with the coat protein gene of PNRSV.The detection sensitivity of IC-RT-PCR is identical to that of RT-PCR.While IC-RT-PCR could detect PNRSV-infected samples more widely, especially from plant samples with low virus concentration in different tissues. And the detection sensitivity of IC-RT-PCR is 1000 times higher than that of DAS-ELISA."

http://en.cnki.com.cn/Article_en/CJFDTotal-XNZK200805025.htm

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A 2004 scientific paper reports on attempts to clean PNRSV infected begonias. From the abstract: "Thermotherapy for 25 days gave 35 and 25% PNRSV-free plants as indexed by DAS-ELISA and RT-PCR, respectively."

From the full paper: "RT-PCR was found to be more reliable technique to detect PNRSV in plants than DAS-ELISA since by RT-PCR, the virus was detected in plants, which were found to be negative for the virus by DAS-ELISA. In biological indexing, i.e. local lesions on C. tetragonoloba, only plants with strong signal in DAS-ELISA gave positive results." Also: "But the number of PNRSV-free plants by thermotherapy alone is much less and about 50 and 37.5% virus-free plants were obtained (as indexed by ELISA and RT-PCR, respectively)."

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" Totally 486 plant materials of sour cherry, sweet cherry, peach, nectarine, apricot and plum trees originating from different stone fruit growing regions of Turkey was investigated for the occurrence of PNRSV in fruit orchards and nurseries. DAS-ELISA and RT-PCR procedures were applied to all samples in order to determine the present viruses. 51 samples were estimated as infected by PNRSV testing by RT-PCR, while DAS-ELISA detected the virus only in 31 samples."

http://www.actahort.org/books/657/657_14.htm

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"Three methods were compared for the detection of prunus necrotic ringspot virus in herbaceous and woody plants: DAS-ELISA, nonisotopic dot-blot hybridization and reverse transcriptional polymerase chain reaction (RT-PCR)."

For PNRSV "Data obtained using herbaceous and woody hosts also revealed that the nonisotopic molecular hybridization technique was 25 times more sensitive than DAS-ELISA and 625 times less sensitive than RT-PCR (Table 1). However, considering the minimum amount of tissue required to give a positive result, the nonisotopic molecular hybridization technique was 500 times more sensitive than DAS-ELISA."

http://ddr.nal.usda.gov/bitstream/10113/35618/1/IND21978494.pdf

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From: The Plant Pathology Journal, volume 27, issue 1, pages 44-52, 2011.

"Comparison of ELISA and mRT-PCR results of 30 samples showed that numbers of infected and mixed infected samples as well as infection and mixed infection rates were significantly higher in RT-PCR (20 and 66.7%) than in ELISA (14 and 46.7%). The results confirm that mRT-PCR is more sensitive than ELISA."

http://www.ppj-online.org/archive.php?a=dv&id=754&vol=27&no=1&cu=

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Thus, part of the "being confident that the virus is gone" depends on using a detection method more sensitive than ELISA.

(Does the additional indexing with herbaceous hosts, Shirofugen Cherry, and/or Multiflora make up for the lack of sensitivity of ELISA? (which 2 virus cleaning laboratories have used in the past or are still using.) From what I have read, I do not think so. Probably the best examination of these methods is found in the 1983 Ph.D. Thesis of P.C.Gardner. If you use the Adobe search function with the keyword "index" you will find his material covering indexing methods. (Because of his copyright restrictions, it appears that I cannot post more detail,))

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Another part of the "being confident that the virus is gone" depends on using a virus elimination procedure more reliable than simple heat treatment and then grafting selected buds on virus free understock. If one runs a Google scholar search using the key words: (virus elimination plant thermotherapy) and limits the search to 2009 and newer, one can see that thermotherapy is being combined with apical meristem culture (with or without an antiviral drug).

It is my understanding (from private e-mail) that at least one major rose testing university program switched several years ago from heat treatment/bud grafting to heat treatment/apical meristem tissue culture for their rose virus cleansing.

The above should be sufficient for the general science familiar reader. Those with advanced science backgrounds may want to continue reading the following:

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1) A number of Austrian papers (by the same laboratory plus other laboratory co-authors) appear to say something which is related to this issue. I purchased one of the most recent of their papers.

Title: Phytosanitary Improvement of Fruit Tree Species: Diagnostic Strategies in Virus-Indexing of In Vitro Plants

Authors: A. da Camara Machado, D. MendonCa, M.S. Lopes, E. Knapp, V. Hanzer, W. Arthofer, H. Katinger, M. Laimer da Camara Machado

Authors affiliation: this was a combined publication from a laboratory in Portugal and a laboratory in Austria.

Published in: Acta Hort., volumn 472, pages 511-516, (1998).

H. Kuska comment: In the actual paper they say that one of the points of the investigation was: "2) Are virus elimination treatments merely reducing the virus titer, but are they failing to eliminate some viruses entirely. If so, how long does it take for those viruses to reaccumulate to detectable levels?"

My comment (H. Kuska) The above sounds like exactly what we are looking for (but not on roses).

In their Results section, they say:

"Furthermore, we tried to understand whether, in some cases, virus elimination treatments merely reduced the virus titer or failed to eliminate some viruses entirely. If so, how long does it take for those viruses to reaccumulate to detectable levels? Depending on this situation, how many tests are necessary to confirm initial negative test results? It is known that elimination treatments may depress the pathogen titer below the threshold level of detection (Fridlund, 1989). In this respect the effects of thermotherapy on the titer of ASGV and ACLSV were investigated in in-vitro-shoots of several apple cultivars. Irrespective of the duration of thermotherapy treatment (depending on heat sensitivity of the plant material), the virus titer of both ASGV and ACLSV initially was decreased dramatically. Shoots that were multiplied in vitro for over 2 years and used as starting material. Before undergoing thermotherapy, they tested 100 % positive for ACLSV, 100 % negative for ApMV and showed values around the threshold level of 36% for ASGV, as shown in Fig. 1 for the Austrian cultivar Maschanzker. After thermotherapy meristems were excised and regenerating shoots submitted to a multiplication step to increase the number of shoots. After 7 months, plantlets were tested again and showed mainly negative values or values around the threshold for ApMVand ACLSV, indicating that the elimination success was satisfactory. On the other hand, after 7 months 7% of the samples were positive when tested for ASGV, indicating a different reaction pattern (Fig. 1)...................................... It is a definite fact that plants with a double infection of ASGV and ACLSV are more difficult to- treat. However, we do not know, so far, on which mechanism this synergistic effect may rely. Samples of the different groups were compared (Fig. 2). From the nontreated positive control plants, only ApMV was not detected after this period in vitro. Values around the threshold were obtained from the originally negative plantlets. In other cultivars, from originally negative clones multiplied in vitro, a high number of samples showed ASGV positive results after more than one year of in vitro culture. ------------------------------------- The detection of ASGV by ITP in shoot tips from several potted plants of Maschanzker grown for 2 years in the greenhouse (data not shown) was, however, a concern. Therefore, the need for a more reliable detection system seems evident."

The DISCUSSION section contained the following: "As it is still common practice in sanitation programs to carry out in-vitro treatment and ex-vitro re-testing for selection of plant material. The result is a lack of knowledge of the speed of recovery of low pathogen levels under in-vitro conditions (IPGRI/FAO, 1994).......................... Elimination treatments depressed virus titer, as could be shown for a wide range of cultivars. Thermotherapy, however, alters or destroys viral proteins, therefore, serodiagnostics are of little value for reliable early screening. Furthermore, there remain the limitations of current ELISA-based serological tests which might be not sensitive enough to detect very low levels of virus. Also, the time required by the different viruses to recover up to a level of detection from low levels of infection will again be dependent on the pathogen host combination. ACLSV was readily detected after re-accumulation above the threshold level whereas the well-known problems in reliable diagnosis of ASGV were further encountered even after several tests of in-vitro cultures (Fuchs et at, 1988, Gilles and Verhoyen, 1992). We assume that molecular diagnostics will improve the aforementioned problems. Other methods which, so far, are not used for routine diagnostics, like PCR or immuno-capture-PCR, will be introduced into the sanitation program to improve the system of diagnosis for in-vitro plants."

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2) A 2008 paper has so many pertinent sections that it is difficult to decide what to include. The following (page 239) is very informative as they actually look at the virus distribution/concentration in the heat treated shoots tips:

"In order to understand why the conventional means of virus elimination described above failed to produce RBDV-free plants, localization of RBDV in shoot tips was carried out. .....revealed strong signals for the virus in almost all tissues...in the non-heat-treated shoot tips...Only the least differentiated cells in the apical dome (AD) of the meristem contained no visible virus signals (Fig. 1A), indicating that these cells contained virus titres below the detection threshold or were virus-free.

The suppressing effect of heat treatment on virus titres was readily observed in the heat-treated shoot tips. ....viral genomic RNA2 ...rapidly decreased and were barely detectable in the shoot tips... following 5 days of heat treatment. Similarly RNA3 were dramatically decreased and only degradation products of the viral RNA were detectable following 8 days of thermotherapy (Fig. 1D). However, the virus was not fully eliminated. Low titres of the viral CP antigen, as judged based on the weak signals, were still detected in most of the new tissues 28 days after thermotherapy (Fig. 1B). ....As the smallest shoot tips (0.1 mm) that could be excised for meristem culture contained LP1, the results of virus localization largely explained why no RBDV-free regenerants were obtained."

The abstract explained that they were able to use a new method (cryotherapy) to eliminate this problem. I should also point out that they utilized Transmission Electron Microscopy to observe the subcellular changes associated with thermotherapy.

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3) So far the emphasis has been on techniques using fast growing apical tips, this 2010 paper indicates that the utilization of root tips may be a superior method.

Title: "Tobacco ringspot virus persists in the shoot apical meristem but not in the root apical meristem of infected tobacco."

(Please click on free preview for more detail.) Tobacco ringspot virus is included in the list of mosaic viruses that are found in roses.

The use of root tips for virus purification is not a new idea. The following gives the abstract to an garlic paper:

The full paper has the following pertinent (to virus elimination) quote in the Introduction: (page 83) "Moreover, root tips may be free from viruses (Pierik, 1987 and the reference therein)....."

The (Pierik, 1987) reference above is to a book:

Author: Pierik RLM

Published in 1987.

Title: In vitro Culture of Higher Plants.

Publisher: Martinus Nijhoff Publishers, Dordrecht

Google link: http://books.google.com/books?id=3DTyQSqa_nAC&dq=Pierik+RLM+(1987)+In+vitro+Culture+of+Higher+Plants.+Martinus&source=gbs_navlinks_s

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My other rose virus sites can be reached from the following index page:

https://sites.google.com/site/rosevirusindexpage/