Do It Yourself (DIY) Test for Garlic Bloat Nematode Andy Leahy <andyleahy@verizon.net> This is a fuller explanation intended to be circulated to inquiring viewers of a YouTube video I posted Dec. 11, 2015, describing a few Quick, Simplified, Do It Yourself (DIY) Home Tests to check garlic intended as seed stock for the presence of the now-infamous, plant-parasitic Stem and Bulb, or “Bloat,” Nematode (Ditylenchus dipsaci). Among United Kingdom allotment gardeners, this pest has been customarily referenced as Onion Eelworm (or Eel Worm). YouTube only allows so many words of description and commentary, and, as usual, I went long. The video itself is here: https://youtu.be/ven5cLVn-Eo These DIY tests assume the grower may lack access to a microscope, but—if that’s not the case—so much the better for use as the capper for any of these procedures. For those without a microscope, we’re going to rely on what is now ordinary personal or household equipment for many people—the key piece being a smartphone with a zoomable camera (though possibly a jeweler’s loupe would also do the trick). The rest of the necessary tools and supplies are simply stuff already in most households, or available at any drugstore. Background In several Northeastern and Midwestern U.S. states and particularly in the Canadian province of Ontario, many Cooperative Extension-type bulletins, web pages, and PDF’s have been recently rendered for commercial, hobby, and home growers—warning of a particular pest that’s currently mounting a re-insurgency against their beloved garlic. Most of these summaries have been put out since a new round of Garlic Bloat Nematode (GBN) infestation was (I believe first) proved by specialists affiliated with Cornell University around 2010. It’s not my plan to rehash all of that knowledge base here, as it’s all Googleable. The bottom line is that affected garlic growers are going to be in quite a pickle for years running forward, fending off this nematode. And there’s going to be an increase in the number of garlic farmers wanting to better know where all of their seed stock stands—because the seed pieces themselves are probably the primary means of transmitting this nematode’s affliction from season to season, from old patch to new patch, and from farm to farm. So questions start to snowball. Most of the official wisdom counsels growers that they need university lab tests to answer these questions, within the confidence level of detectability on the basis of a small sample.

That’s going to be a lot of tests. As an alternative, I have found that, with or without a microscope, much of the first or second pass testing can also be done by amateurs at home. Here's how. Gear and Setup Needed for a First-Pass Shortcut Garlic Slab Test Assuming a microscope or a smartphone with zoomable camera is handy, gather a single-sided razor blade or ultra fine knife, chlorine-free water (not tap water from a municipal system, but rather either distilled, pretty sure most bottled waters should be okay, spring water, or untreated household well water), some ordinary drugstore 3% strength hydrogen peroxide, and some small pieces of clear flat glass. Choose among the most suspicious-looking heads of garlic—say three or four of them—and after breaking out the cloves and taking a look inside past the clove skins, select one of the sorrier-looking cloves from each head. As I did much of my testing in November and December 2015 on hardneck garlic that was harvested in late July or early August that same year, I can report that the prime suspects in cloves aged four months since harvest will be the ones that by then feature significant areas of light-brown-darkened, seemingly translucent flesh, found to extend well inward upon slicing, and often more highly concentrated toward the growing tip end. The bad color subtly distinguishes itself from a healthier clove’s opaque brightness, which is really more the shade of vanilla ice cream than straight white. Not all that diagnostic are discrete patches of dried brown flesh, mostly limited to the surface of the clove, which I believe is fairly common evidence of some prior, larger scale wound. On the other hand, for seriously brown-darkened and withered cloves, there are likely to be nematode carcasses and egg stages in there by the thousands, but—four months after harvest—it’s possible that very few of these will be still alive or active in order to convincingly help tell the tale, especially without a microscope. While it’s not strictly necessary to peel each of these sample cloves (and viewers can see from my video stills that I didn’t always bother to do so), I’m going to recommend peeling in order to help fend off the possibility of reaching a false positive—due to finding representatives of some other nematode species that were simply riding in some dirt on the clove skin, head cover skin, or roots. We are identifying Garlic Bloat Nematodes here not because we know precisely what they look like (which we don’t, compared to the colossal universe of cousin nematode species out there, 24/7/365, munching away in every corner of this world), but simply because of the food matter they had to have come out of.

A First-Pass Shortcut Garlic Slab Test Take from each sample clove a few slabs of flesh, cut as thin as practical using a single-sided razor blade or ultra fine knife. I have found that a section cut lengthwise from the clove offers more surface area for the next step of soaking, but I suspect many nematodes, if on hand, can survive the knife and find an exit from a slice cut out of the

clove in any orientation. Arrange these thin garlic slab samples on the clear flat glass. Soak with drops of chlorine-free water, and then, counter-intuitively, add drops of 3% hydrogen peroxide. The now somewhat further diluted H2O2 should spread all over the tops of the garlic slabs and eventually get underneath, possibly even floating each piece a bit on the bubbly liquid. I had good luck letting it bubble away for about 10, 20, or 30 minutes before moving on to the next step. (Ordinarily, hydrogen peroxide is considered to be a worthy disinfectant, and I have often used it on skin wounds—pleased to experience the visual feedback loop of all that foaming goodness, and convinced by the folklore of my upbringing that that’s the Bad Stuff getting crucified, or something. Now I’m not so sure, having personally witnessed Garlic Bloat Nematodes energetically swimming with seeming joy in shallow pools of this suddenly oxygenated environment.) After maybe 20 minutes, towel off some of the excess fluid, especially from the undersides of the glass slides, and get ready to take a look. With access to a microscope, one can go directly to town with these samples, paying particular attention to the perimeter of each garlic slab, where a moat of peroxide bubbles gives way to areas of clear, relatively flat fluid. Without a microscope, there are two views to check using a zoomable smartphone camera—the first with the garlic slabs still sitting on the glass, and the second after getting the garlic out of the way. Setup for Using a Smartphone with a Zoomable Camera Without a microscope, one must jerry-rig some kind of moveable kitchen counter platform with a black background placed directly underneath the glass slide—maybe black construction paper, maybe black cloth, maybe something mostly flat in black ceramic, or possibly even black plastic. This black background platform should be high enough up off a counter so that the glass slide will sit about 3 inches below, say, a cabinet shelf. That shelf will be where a smartphone with a zoomable camera can be laid down and kept steady, aiming downward, with the lens sticking out over the shelf edge. My setup for a microscope-less situation was an iPhone 5 looking down from a kitchen cabinet shelf upon my glass slide, perched atop the bottom of an overturned black coffee mug (held up at about the right height by an overturned, standing flashlight). Sometimes, I used a piece of black construction paper as the backdrop. One key element shows subtly in some of my slideshow stills from the video: Ignoring the camera flash necessary for taking these photos, note that most of the light on the sample slide is coming from behind and from very slightly above. This was due to the happenstance of my having under-counter fluorescent tube lights. But, if that’s not available, one can get by with a flashlight aimed sideways at the sample in a darkened room. I’m not going to be able to supply smartphone camera operation tips here for every possible model, but let me just recap what I did: 1) Again, this is an iPhone 5, which is currently one or several generations back, depending on how the count should go; 2) Poking around online, I’m so far unable to ascertain the power of the iPhone 5’s zoom, but it’s probably just a digital crop thing (at maybe 5X?), not a true optical zoom that actually gains better resolution; 3) I haven’t run out (yet) and bought any external stick-on macro or zoom lens attachments to further enhance my

view, but I have been sorely tempted by the ever-increasing variety of third-party offerings and by the ever-dropping prices; and 4) Once zoomed in, there was a learning curve to figure out how to force the camera to find and lock itself in on focal and exposure settings that were just right for revealing the nematodes against a black backdrop. Again, the perimeter of every bubbly garlic slab is the place to start looking. There’s not enough magnification to be certain of distinguishing a handful of dead or dormant nematodes from all the other possible filaments that might be suspended and glowing in this light. But spotting a large number of suspicious, unmoving filaments could count as a positive test result—from a colony that may have eaten itself into rot and ruin. Better yet is finding even just a single live nematode. If there happen to be any there, swimming in the clear, that will be a thunderbolt to most garlic growers, answering a lot of questions about past losses, and setting the stage for future struggles against this now-known pest. I have found—even with the bubbly garlic slabs still obscuring most of the actual action hiding on the slide—that a microscope will have no trouble showing a number of nematodes writhing partly under the ring of peroxide bubbles. With just a fully zoomed smartphone camera to work with, however, there might be only 1-3 live nematodes (out of 100-300 actually present) that have, for some reason, swum off on their own, well into the dark zone—far enough away from the bubbles and that delicious schmear of garlic juice to be unquestionably clear while viewing the action live through a smartphone’s display. The best precaution against missing any swimmers is to briefly slide around the chunks of garlic, then remove them, and look again. That can be like taking the hat off of Hades. To eliminate any further doubt, make a short video of the scene and then replay it later on a desktop computer, full screen.

Second-Pass, Modified, Simplified, Thoroughly Home-Brewed, Baermann Funnel If there is no slam-dunk positive using the first-pass shortcut test described above, we can proceed with prepping the rest of the sample garlic cloves for a second-pass test. Further gear warranted: a blender, an ordinary wire mesh kitchen sieve, a bowl that’s sized somewhat larger than the sieve, and something to suck up liquid with and later mete out droplets (an eye dropper, a hypodermic syringe without the needle, maybe a turkey baster, or the rubber ball warm water flusher thingie from an ear wax removal kit which I’ve had quite a bit of luck with). Chunk up each clove just enough so that a blender can get a bite on the pieces, suspended in chlorine-free water. With the blender, we’re certainly not looking for anything remotely close to a puree, but I’m hard-pressed to describe exactly what’s best, and I’m not sure it matters all that much. Maybe no more than a total of 10 seconds of intermittent chopping on the slowest setting? The history of this technique dates from 1917, which is probably just the year of publication of a paper from Dutch physician G. Baermann. While working overseas in Java, Dr. Baermann was the first to have and report a blazing insight into solving the nettlesome problem of separating nematodes from the clutter of their soil or plant host material (or possibly even, for all I know, from animal flesh). Baermann’s solution was to simply encourage the buggers to swim free of whatever comfy bailiwick they are well-adapted for, and then to give them some time to gradually sink downward in the water due to their own slightly heavier weight. The process has since been shorthand-referenced thousands of times by nematode scientists worldwide, usually as the Baermann Funnel, or the Baermann Process, or any number of simplifications or modifications which still pay credit to his original scheme. All of them work according to same principles: They increase the chances of finding live nematodes by first asking them to swim free in water and then allowing time, gravity, and the smooth inside surfaces of the containers to concentrate them down low at a suitable collection point. Our thoroughly home-brewed Baermann Funnel consists simply of an ordinary kitchen sieve perched along the rim of a smooth ceramic or metal bowl. Both sieve and bowl should be particularly chosen because we want a bowl that’s just deep enough so that there will be an inch or so of clearance between the bottom of the bowl and the bottom of the sieve. Dump the blender contents into the sieve and let the water drain through. If it turns out there’s still not enough liquid to keep the pile of chopped garlic thoroughly wet against all sections of the sieve screen, add some more chlorine-free water to the blender, slosh it around to catch any remaining garlic bits, and dump it into the sieve. Or just add some more chlorine-free water straight over the top. After that, we’re again going to take the counter-intuitive step of prolonging the period of liveliness of any nematodes that may be hiding in the garlic by oxygenating the water with occasional bursts of hydrogen peroxide. I have tried to add just enough hydrogen peroxide to let it foam up slightly, but it often turns out to be too much, causing a mass of garlic chunks to ride a cloud of foam, soon cresting over my sieve and bowl. Probably we’re better off taking it easy on the H2O2 in an effort to prevent a large supply of oversized garlic chunks from polluting the preferably mostly clearish water in the bowl, or to avoid making a bigger mess in the kitchen than necessary. After maybe 10, 20, or 30 minutes, try the first check on this second pass. Get that suction device handy. Lift the

sieve up out of the way temporarily, and collect a full suction's worth of liquid from the very bottom of the bowl. Again, both live and dead nematodes are known to be heavier than water and to therefore wind up more highly concentrated at the bottom. Though it’s better to wait a bit to let any nematodes resettle after the tsunami caused by our sample collection, there’s little to be lost in going directly with droplets to a fresh glass slide for a microscope or smartphone test. If there’s no there there at first, our chances of detecting any nematodes can be easily improved in follow-up looks. Between tests, prop up the filled-up suction tool with the opening at the bottom, so there’s more time for the nematodes to swim and sink in a direction that’s convenient for our purposes—to the drip point of our suction device. I have found that the number of swimmers can increase even 10 minutes later, and the numbers even seem to drop from higher to lower between the first and last droplets separately placed in a series onto the slide. Note also that this is just a first try after giving any prospective, especially ambitious nematodes only a short time to exit their garlic waterfront condos and make it all the way down to the bottom of the little kitchen swimming pool we’ve set up for them. That’s probably equivalent to impatiently expecting a human scuba diver to swim down 1,000 feet in the ocean in just 20 minutes. So, hours or even a day or so later, flush out the suction device and return for a fresh sample from the very bottom of the bowl, left sitting underneath the perched sieve of soaking garlic. The odds of reaching a result to be sure of improve with every check—either the joy of seeing absolutely no nematodes, or the heartache of finding even just one of them. If testing goes on for some time, and the peroxide bubbles look like they’re dying down, add some more.

Some Closing Notes My outreach is partly inspired by the observation that many garlic growers—already fearing the agony of knowing the worst—may be finding further motivation to procrastinate actual professional testing due to the fees, the shipping expenses, and the wait times of sending a sample to a university lab. The lab-testing costs go higher in order to learn where even a modestly compulsive garlic operation stands in every possible situation, from year to year. And some may still possibly be in full-scale psychological denial as to whether Garlic Bloat Nematode may in fact be—beyond the legions of other, viral, fungal, or bacterial possibilities—the primary culprit behind their ever-increasing losses in the field. Others remain in a state of worrisome ignorance, partly because GBN symptoms in the field too often look a lot like so many other possibilities (some of which are, in fact, present and even visually dominant—ushered in by nematode damage inflicted upon the plants during the first seating). Even with relatively high nematode loading, a good garlic farmer can still go year after year producing some surprisingly great looking bulbs, with only a tolerable portion mysteriously lost. That was the case with my own garlic group, up until a seed buyer got back to us in late Fall 2015 with some bad news from a lab test he had independently commissioned prior to his plant-out—against our own garlic! Ever since—knowing how much garlic we have sold over that previous five years (or had just got done selling that very Fall) “for food or seed”—we have been reeling with the shame of Typhoid Mary, who only now suddenly knows the truth. There’s a lot of misplaced motivational pressure to just stay quiet about it, to try and work it out alone, and to flinch from participating in the informational exchange—for fear of freaking everybody out, including food garlic customers. When confirmed and magnified, up close and personal, GBN is unquestionably icky to contemplate for anybody that’s been eating that garlic all along. But the facts are that these nematodes are not actually a health

issue for garlic eaters. These nematodes are incapable of surviving cooking, and are not adapted to live through the process of human digestion of even raw-eaten cloves. Even if they did survive, they’re not built to feed on animal flesh. Instead, GBN is a thorny issue for all future production. Like venereal diseases, Garlic Bloat Nematode may be at the point of an outbreak among garlic growers partly because these members of the unsegmented roundworm grouping are assisted both by human behavior and by the very nature of their niche. Garlic has been for hundreds of human generations grown exclusively from fleshy cloves naturally cloned from the mother plant (rather than from tiny, sexually reproduced, true botanical seed, which can be cultivated from some garlic varieties only with great difficultly, though this is now increasingly available among plant breeding hobbyists). There just happens to be lots of comfy, cozy, wet, flavorful, energy-laden, possibly even frost-resistant room inside all of those cloves, allowing thousands of nematodes to simply hang out in just the right place until humans do them the solid of re-planting in the Fall—possibly right smack dab within next Spring’s smorgasbord of previously uninfected ground. Or they might wind up traveling hundreds or thousands of miles across state or national lines to a brand new free lunch spot. We garlic enthusiasts do enjoy buying, selling, and swapping new varieties, but there has not been enough thought given to this plant’s inherent dangers in engaging in such trafficking. Few welcome the trouble and expense of another lab test to protect ourselves before planting out every new acquisition, but I’m hoping many more garlic buffs can learn to do their own checking. All agree that there is no absolutely reliable negative test result for GBN, and I certainly don’t claim to have rolled back that limitation. Even if Cornell or some other professional lab issues a negative result, all that means is that their specialists couldn’t find any nematodes in that sample using their methods (which, to be fair, are known to be the best-possible means of detection). But all it takes is one male and one female, lurking somewhere within the flesh of a planted clove, and all bets are off in future years for the health of even a garlic patch newly installed on virgin ground, and all seed stock reared from that patch. Somewhere I read that this nematode’s tendency to make itself a pest against allium crops has been the subject of Northeastern U.S. academic reportage since at least the 1930s, but back then it was primarily the onion farmers who were suffering. The onion guys broke the cycle by transitioning to new soil and then sowing their crop from tiny, pest-free, true botanical seed, rather than keeping down the path of ruin by continuing to plant cloned onion sets. For garlic growers, it will not be as easy or as low cost to transition everybody’s grow-out away from the traditional, cloned seed and toward true seed sources (which will actually, more precisely, most likely, be implemented as clones grown out over several generations in a lab or greenhouse setting and then to virgin soil – but all of it originating from first-year true seed gene stock). Many of the old, cherished, named varieties might be abandoned in favor of a range of newly created, newly named FMO’s (Farmer Modified Organisms), but that could be where we’re headed.