Annual CyMV/ORSV testing

I first became aware of orchid viruses in the Summer of 2005 while attending a meeting at the Golden Gate branch of the Cymbidium Society of America. I remember Paul Chim discussing/demonstrating how to divide and re-pot Cyms to prevent infection and spread of orchid viruses, in particular, the two most common orchid viruses Cymbidium Mosaic Virus (CyMV) and Odontoglossum Ringspot Virus (ORSV). I had never heard of orchid viruses, so this caught my attention. A month or so after the meeting, I sent off leaf cuttings to Critter Creek Laboratory to test my Cym collection (at that time, it was only 15 plants or so) for CyMV and ORSV. The test results stated that some of the Cyms were infected, so I then realized that learning to how to manage orchid virus pathogens, in addition to other pathogens such as mites, scale, mealybugs, thrips, etc…, was going to be a real issue for me if I wanted to continue growing orchids.

The AOS has been regularly publishing articles about orchid viruses about once a year since 1987, so this topic isn’t new to the orchid community as a whole—only to me. I later found copies of Roger Lawson’s article “Orchid Viruses and Their Control” (AOS Handbook on Orchid Pests and Diseases, pgs 66-101, revised edition, 1990) and Gail Wisler’s book How to Control Orchid Viruses: The Complete Guidebook (Maupin House Publishers, 1989). There are plenty of other resources to be found on the internet discussing how to control orchid viruses.

Why should one be concerned with orchid viruses? Well, virus-infected plants may present with undesirable features such as necrotic spots along the leaves, decreased flowering capacity, smaller than expected flowers and/or color breaks. Most people are familiar with harmful orchid pathogens such as insects, fungi and bacteria, and there is an abundance of products on the market to control or eradicate these disease vectors. However, no such technology yet exists to eradicate virus infection in orchids (or humans). Therefore, the existence of a virus-infected orchid in one’s collection presents a never-ending risk of spreading the incurable pathogen to non-infected plants

Improving my orchid husbandry had to take a backseat for over a year because I changed careers, moving twice within six months, including across the country. Once I got settled into the new house and on top of my workload at the office, I could then afford more attention to the Cym collection, which was growing in size from having purchased seedlings and mature plants from various persons. While surfing the internet, I had found a company, Agdia Inc., that sells test strips in kit form so that one can test one’s orchids at home for CyMV and ORSV infection. It was near the end of the calendar year, so New Year’s resolutions were in order, and mine was to re-visit CyMV/ORSV infections and test my orchid collection.

Below is a pic of two samples doubly infected with CyMV and ORSV (the top red bar is the control line to let you know that the test is working; the middle red bar indicates ORSV infection; the lower red bar indicates CyMV infection).

The results were not good. I had, unfortunately, achieved a CyMV and/or ORSV infection rate of 35%! I was stunned, upset and disappointed with myself. Nothing to be proud about here. The infected plants were immediately thrown away, including a number of my favorite Cym hybrids in bloom with gorgeous flowers. The economic loss was also jarring. I needed to seriously reconsider how I was growing my Cyms.

One of my colleagues at our local orchid society consoled me with the following: “I believe that the U of [name deleted] tested their entire crop of orchids, found a 94% infection rate. Discarded the bunch and restarted with fresh plants tested on the way in. Found a 6% infection rate in the crop the next year, discarded them and from my reading the results of their virus free orchid collection is apparently very apparent in robust plant growth and inflorescence production flower counts.”

From this hopeful anecdote, I embarked upon the following practice: i) test all stock annually to detect infected orchids that had converted over the previous year, and ii) test all incoming orchids to prevent the introduction of diseased stock. Within three years, per the previously observed 94% to 6% to 0% rate, I should hope to obtain a CyMV/ORSV-free collection.

With this in mind, I paid closer attention to how I handled the orchids at all stages and how I might be promoting the transmission of virus from one orchid to another. In hindsight, I think a significant reason for the high infection rate was because I had placed multiple Cyms in the same water catchtrays as a matter of expediency when packing, moving, unpacking, packing, moving, etc… This was a fatal error because the runoff of an infected plant could easily expose another plant to the viruses. So, I now have each Cym with its own catchtray. Other changes include: i) use virgin razor blades when grooming and/or dividing each orchid, ii) use virgin flower stakes (bamboo is cheap), iii) use virgin latex or nitrile gloves and newspaper when dividing and re-potting each orchid, and iv) pay attention to insect control to mitigate this potential vector from spreading virus from one plant to another. I still re-use some plastic pots and plastic catchtrays. I clean them off with a scrub pad, soak them in bleach (1 cup bleach per gallon water) for at least 24 hours (a matter of arbitrary convenience), and then rinse off the residual bleach. While this practice provides a source of vulnerability, I have not yet observed this to be a causal agent for virus infection. I know that some in the trade never re-use plastic and simply buy virgin pots, and I may well be converting over to that practice soon enough.

One year after the first cull of CyMV/ORSV infected plants, I re-tested those that had previously tested clean. 7% had converted positive. While not great, the good news was that not ALL of my Cyms from the previous year’s testing had become infected. Furthermore, the orchids that I had acquired during the year that had tested clean upon receipt did not re-test as infected. Re-testing is important because it may take as many as 30-90 days for a newly infected plant to culture enough virus particles to be detected using the kit. The infected plants were thrown away, of course.

This weekend, two years after the first cull of CyMV/ORSV infected plants, I re-tested all of my orchids, including those that had tested clean the previous year. I was pleased to find that 0% tested CyMV/ORSV positive. Furthermore, the orchids that I had acquired during this last year that had tested clean upon receipt did not re-test as infected. It appears that by practicing steps (i) and (ii) per the University’s example, I have achieved a 35% to 7% to 0% infection rate.

Notes on Agdia Immunostrips

I’ve observed sometimes that the Immunostrips may yield VERY WEAK, double-positive signals, almost like “ghosts”. I don’t have a firm explanation for such a result. I would post a picture of such ghosts, but they’re too faint and difficult to photograph without a macro lens (which I don’t have). If you see these ghosts appear on the Immunostrip, then do NOT throw the plant away outright. I found that when I re-tested the plant(s) (nonetheless quarantined from the rest of my collection) at one, three and/or six months after seeing the ghosts, they tested negative. If the cause of the ghosts were an early stage of infection, then I would expect to see stronger signals at the later timepoints. But this was not observed. Instead, an alternative explanation may be that the test strips that yielded ghosts were past their expiration date. My orchid society colleague had noticed that ghosts appeared if the tissue sample was too large, implying that too much plant tissue adversely affects the performance of the test. His experience is also that for Bulbophyllums, one should use root tissue instead of leaf tissue because it appears there is too high of a polysaccharide content in Bubo's leaves which will cause the test to fail.

Elliot et al (AGRIS record 80(10):1160-1164, 1996, excerpted from Abstract) teaches that “Several orchid plants gave ELISA reactions greater than three times the negative control with all the virus antisera tested. Other methods did not confirm the presence of virus in these plants, however. Indeed, several preimmune sera also reacted with some of these plants. Caution must be used in interpretation of low ELISA values even when these reactions are clearly greater than those of uninfected controls. These results illustrate the need to utilize more than one diagnostic technique before discarding a valuable orchid plant.”

One can, for example, send a leaf sample to Agdia for direct testing of CyMV/ORSV and other orchid viruses via their in-house immunological and/or nucleic acid testing means. My colleague’s orchids that yielded “ghosts” in the home environment tested negative via Agdia’s in-house services.

“Constant Vigilance”

(Captain Jean Luc Picard, The Drumhead, Star Trek: The Next Generation, Season 4, Episode 21)

Do I think I have an orchid collection free [emphasis added] of CyMV/ORSV? Absolutely not. I’ve been a scientist for too long to be deceived by this value. Viruses are microscopic pathogens, so they could well be lurking in and around my growing areas without my knowledge, some place I’ve missed, etc… There may also be an infected plant that is producing too few virus particles to be detected by the Immunostrips, but at a later point in time, due to one stressor or another, will develop a robust infection and virus particle production. However, I do think that the overall health of my orchid collection is improving because of these efforts to eradicate the clearly infected plants and to prevent virus spread between plants. So, I’ll still continue to test each orchid for CyMV/ORSV infection before it enters my collection, as well as before I re-pot and/or divide it.

Time to enjoy a glass of wine to celebrate the minor victory…