Discussion of whether virus have been found to return to heat treated plants

Discussion of whether viruses have been found to return to heat treated plants.

In the following quotes, when something appears in bold print, it does not mean that it appeared that way in the original; the bold print was added by me (Henry Kuska) for emphasis. This page gives the information that I have collected from my own literature searches and from others posting on the internet. Please let me know if you feel anything is not clear or is not addressed at all as I am continually updating/modifying it as I get feedback.

The following was stated recently 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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The following statement was made in 1988: "Well, Malcolm, then you had better speak with the folks at Davis!
Because some years ago they reported to the group at the inaugural meeting of a California rose plant grower's association meeting 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.
http://groups.google.com/group/rec.gardens.roses/msg/61458eaadecee942?dmode=source
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Is there an independent statement that supports what happened at Davis?
The following link contains the following 2005 statement: " 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
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THUS, the original Davis statement has been confirmed. 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.

Although this article does 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."
A QUOTE FROM:
Title: Optimizing detection and management of virus diseases of plants
Author: 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 92521

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"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."

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."
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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.).

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:

"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."

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)."

" 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."

"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."

"Comparison of ELSA 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."

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

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:

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.

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).

"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.

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.

(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:

In the Introduction: (page 83) "Moreover, root tips may be free from viruses (Pierik, 1987 and the reference therein)....."

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H. Kuska comments. What I stated in 2005 "It now "appears" (to me) that a combination of heat treatment and / or antiviral drug and tissue culture may "outrun" the virus. Notice I say "may" because this is a new field and there simply is too much that apparently is not yet understood/known to make definite statements. " can now be changed to:

It appears that the field has advanced to the point where scientists can be confident that micro sized growing tips (including root tips) meristem tissue culture techniques have been developed to "outrun" the virus (see 2009 Google Scholar search referred to earlier in this thread). Plus there are now highly sensitive (PCR and electron microscope) detection systems available to guard against the possibility that the virus concentration is not zero but just below the ELISA detection limits.