Easy On-Site Tests for
Fungi and Viruses in Nurseries
and Greenhouses
JOHN N. KABASHIMA, Ornamental Horticulture Farm Advisor, UC Cooperative
Extension, Orange County; JAMES D. MACDONALD, Professor and Plant Pathologist,
Department of Plant Pathology and Agricultural Experiment Station, UC Davis; STEVE H.
DREISTADT, Senior Writer, IPM Education and Publications, Statewide IPM Project,
UC Davis; and DIANE E. ULLMAN, Professor, Department of Entomology, UC Davis.
To manage a disease effectively, you often have to determine which specificpathogens are present. Many control actions are effective only against certain
pathogens; if you do not correctly identify the cause of unhealthy plants, your con-
trol actions may be ineffective, resulting in plant damage and wasting time and
money. Fortunately, easy-to-use test kits can help you determine whether or not cer-
tain plant pathogens are present so you can make an informed decision. This publi-
cation describes test kits that identify some common plant viruses and root and
crown decay fungi. You can use kits to test for certain viruses infecting aboveground
parts of virtually any plant. Some kits that detect root-infecting fungi are recom-
mended for greenhouse and container-grown nursery plants. Using special proce-
dures not discussed here, you can also use these kits to test for the same fungi in
water and container media. The Phytophthora test kit has also been used in field soil
assays and tests of field crops, but neither of these uses is currently recommended.
R O O T A N D C R O W N D E C AY F U N G I
Phytophthora, Pythium, and Rhizoctonia are common root and crown decay fungi
that affect many different plants. Dull, yellow, or wilted leaves are often the first
symptoms noticed in plants affected by these fungi. Infected plants become stunted
and die when their roots and crowns decay. Similar symptoms are caused by other
pathogens, insects, and noninfectious disorders, so specific diagnosis can be difficult.
Fungal fruiting bodies such as spores and sclerotia are used to identify many
pathogens, but Phytophthora and Pythium species produce no fruiting bodies visible
to the naked eye. Rhizoctonia may form coarse weblike growths around rotted basal
stems, but the tan-white fungal mycelia are neither consistently present nor reliably
indicative, and other fungal species also produce whitish mycelia. In the past, to
confirm the specific fungal species present you would have to send a sample of sus-
pect tissue to a plant pathologist who would culture it on laboratory media and
examine it under a microscope. The process would typically take 1 to 3 weeks.
T O S P O V I R U S E S
Tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV) are
important viruses spread by thrips. These viruses in the genus Tospovirus have wide,
overlapping host ranges, and damage many agricultural and ornamental crops.
Tospoviruses produce a broad range of symptoms, including stunting, yellow or
white spotting, black or brown stem or leaf necrosis, ringspots, defoliation, vein
necrosis, and dark or yellow line patterns, mottling, or mosaic.
P U B L I C A T I O N 8 0 0 2
UNIVERSITY OF
CALIFORNIA
Division of Agriculture
and Natural Resources
http://danrcs.ucdavis.edu
http://commserv.ucdavis.edu/freepub/
For field diagnosis of viruses, growers commonly rely on recognition of damage
symptoms, consultation of published lists of host plants, and knowledge of local dis-
ease history and how specific viruses develop and spread. Visual diagnosis of virus-
es, however, is often inaccurate. Symptoms vary by cultivar. Some plants serve as
hosts for many different viruses. Multiple viruses can occur in a plant at the same
time. Viral symptoms can easily be confused with the symptoms of nutritional dis-
orders, herbicide damage, or diseases caused by bacteria or fungi. Some infected
plants can be symptom-free for many weeks, showing symptoms only when the crop
matures. Symptom-free plants can be a source of infection for nearby crops.
Electron microscope analysis is the traditional method for expert diagnosis of
viruses. However, electron microscopes are expensive, results take at least several
days, similar viruses often cannot be microscopically distinguished, and few diag-
nosticians are available to perform this work. You can also use plant indicator series
or bioassays to diagnose viruses, either by rubbing a solution from symptomatic
plant tissue onto different indicator plants known to develop specific symptoms in
reaction to certain viruses or by using their insect vectors for transmission. Indicator
series are relatively slow, and necessitate the constant production of the indicator
plants, and sometimes the insect vectors, that the bioassays require.
PAT H O G E N T E S T K I T S
Now, instead of sending samples to a laboratory and waiting for answers, you can
use quick and relatively simple, commercially available test kits to identify certain
pathogens. Most of these field-useable test kits employ a serological technique called
ELISA (enzyme-linked immunosorbent assay). This technology is routinely used in
home pregnancy tests, blood tests for AIDS and hepatitis, and sampling for pesticide
residues. Fungal test kits, sold under the brand name Alert, can detect Phytophthora,
Pythium, and Rhizoctonia. The QTA-Tospo brand test kits can diagnose impatiens
necrotic spot virus and tomato spotted wilt virus. Other test kits are available that
detect other fungi, certain bacteria (e.g., pathovars of Xanthomonas campestris), and
many viruses, and new kits are constantly being introduced. Contact your local
Cooperative Extension advisor or product suppliers for the latest information on
pathogen test kits.
A separate kit is available for each fungal genus and each tospovirus. You can
run multiple tests at the same time. Each kit contains all necessary materials, includ-
ing detector devices sensitized with antibodies that recognize and react with pro-
teins from the target plant pathogen. Some test kits require refrigeration and must
be used before an expiration date printed on the packaging.
U S I N G T H E E L I S A T E S T K I T S
Test kits for Phytophthora, Pythium, and Rhizoctonia fungi can confirm pathogen
presence in as little as 10 minutes. First you collect tissue samples from the suspect
plants, then you macerate them by grinding between abrasive pads, and treat them
as shown in figure 1. After you complete the sample treatment procedure, a visible
color-change reaction in the detector device will indicate infection. A “positive con-
trol” dot will darken to indicate that the test kit is working properly. If the target
fungus is present in the sample, the “pathogen detection” dot will also darken. If
the pathogen detection dot does not darken, the target fungus is not present in the
plant sample.
EASY ON-SITE TESTS FOR FUNGI AND VIRUSES IN NURSERIES AND GREENHOUSES 2
The virus test kits require more skill and patience than the root decay test kits.
Virus kits must be used at a clean workbench area with running water. Samples must
be set aside for hour-long incubation periods between certain steps (figure 2).
Phytophthora and Pythium Test Kits
The Alert test kit for Phytophthora has been used to detect all common Phytophthora
species. Pathogen detection can be accomplished when as little as 0.5 percent of a
plant’s roots are infected. The Phytophthora test does not react with other common
soilborne fungi, including species of Fusarium, Mortierella, Trichoderma, Rhizoct-
onia, Rhizopus, and Sclerotinia.
Exercise caution when interpreting test results. Pestalotia spp. may react weakly
with the Phytophthora kit. These fungi mostly cause blight (spotting, dieback, and
EASY ON-SITE TESTS FOR FUNGI AND VIRUSES IN NURSERIES AND GREENHOUSES 3
Figure 1. Tests for Phytophthora, Pythium, and Rhizoctonia root and crown decay fungi can be performed on-site
by growers in about 10 minutes: (A) collect and grind samples using abrasive pads; (B) fold pads and insert them
into the extraction solution; (C) apply solutions to detector; (D) examine detector dots for color change. Illustrated
here is the Alert root decay test kit from Neogen Corporation.
2 3 4 5
A.
Figure 2. On-site plant virus test kits for tomato spotted wilt and impatiens necrotic spot provide results within
several hours. The methods for use are detailed in information from the suppliers, and are summarized here: (A)
place symptomatic plant tissue into the mesh-lined pouch provided with the kit and crush the sample by placing
the pouch on a hard surface and rubbing it with the blunt end of a pen; (B) insert a pipette-like plastic straw into
the pouch and collect liquefied plant material; (C) place sample drops into a plastic well, which is on a strip with
other wells for additional samples and controls without liquid to help ensure accurate results; (D) place the plastic
well strip into a container with a moistened paper towel and allow the samples time to react with a coating on
the inside of the wells; (E) after 1 hour, rinse the wells with water, then (F) add special solution and place the well
strip into the moist container for another hour. Repeat this last rinse, fluid addition, and wait procedure; (G) after
30 to 60 minutes, if the target virus is present, a visible color change occurs in the test wells. Illustrated here is a
QTA-Tospo kit test from Agdia, Inc.
D.C.B.
A.
B.
C.
D. E. F. G.
decay) of aboveground parts of trees, woody shrubs, and vines, so you may need to
use characteristic damage symptoms, plant parts affected, and host lists to help you
distinguish Pestalotia spp. from root decay fungi.
Pythium and Phytophthora are closely related fungi, and at high concentrations
they can cross-react (i.e., Pythium may cause a positive reaction on a Phytophthora
kit, and vice versa). In fact, a few Pythium species do not react at all with the Pythium
kit and react only with the Phytophthora kit. We recommend running both tests on
the same samples because the tests complement each other. These cross-reactions
generally are not a problem, since cultural and chemical controls are the same for
Phytophthora and Pythium. If some sort of regulatory decision is involved, however,
you may require more-exacting follow-up tests.
Rhizoctonia Test Kit
Use the greatest caution with the Rhizoctonia test. Rhizoctonia solani is an aggressive
pathogen, but many other Rhizoctonia species are not. Some Rhizoctonia species are
beneficial mycorrhizal fungi, and many are simply common soil fungi involved with
the decomposition of organic matter. The Alert Rhizoctonia test does not currently
distinguish between R. solani and other Rhizoctonia species. Since nonpathogenic
types may colonize fine roots, complete reliance on this test could result in a mis-
taken diagnosis.
Consider limiting use of the Rhizoctonia test to bench-grown crops, including
most greenhouse and shadehouse plants. Use kits on plants less than a year old that
have not contacted unsterilized soil, since these plants are less likely to be colonized
by nonpathogenic Rhizoctonia spp. Limit tissue sampling to lower stem and crown
tissues to avoid the fine roots that can be colonized by nonpathogenic Rhizoctonia spp.
Collecting Root Decay Samples
Good sampling technique is critical to effective use and interpretation of ELISA
tests. Strive to collect only tissues that have recently been infected by the primary
decay pathogen. By taking samples from the leading edge of an infection next to
healthy tissue, you maximize the possibility of detecting the primary decay
pathogen. A test that involves severely rotted tissue is usually a waste of time and
test kits.
Remove the test plant from its container and examine the root system to deter-
mine which part of the plant to sample. Start by examining for necrosis in the lower
stem and crown area. If this area is healthy, knock or wash away some soil to expose
major roots and examine these for necrosis. Keep working down until you detect
transitional (healthy–infected) tissue, and then collect a sample for testing that
includes both apparently healthy tissue and adjoining necrotic tissue. When you are
testing for decay fungi, your most reliable results will generally come from sampling
infected crown or major root tissue. Always test more than one plant using separate
kits before you base treatment actions on test results. Wash your hands thoroughly
or wear and discard disposable gloves after handling diseased plants to avoid spread-
ing plant pathogens on hands.
W H E N T O U S E T E S T K I T S
ELISA test kits reliably determine whether target pathogens are present. Use the kits
to test plants whenever you suspect a pathogen infection. The test kit is only one
tool in an overall disease monitoring program, but test kits can be highly cost effec-
tive, compared to the costs of unnecessary or wrong treatments, late control actions,
or damaged crops.
EASY ON-SITE TESTS FOR FUNGI AND VIRUSES IN NURSERIES AND GREENHOUSES 4
Besides diagnosing the cause of poor plant health, on-site test kits are useful for
regular monitoring for viruses in symptom-free plants of susceptible crops. You
might also consider using root decay test kits as part of routine monitoring, so long
as you practice good sampling methods and observe the cautions presented here. In
particular, you might consider a regular testing program for “mother blocks” and
areas where cuttings or other new plants are received or shipped out for propaga-
tion. Routine testing can be especially useful when plants are young; most crops are
more severely affected by pathogens if plants are infected during early growth.
Be aware that negative results in a specific ELISA test do not rule out the possi-
bility that another pathogen is causing disease. You can also get negative results if
you sample and test the wrong tissue. When testing for viruses, sample the youngest
symptomatic leaves. When testing for decay fungi, collect samples from the leading
edge of an infection, where apparently healthy tissue and infected tissue meet.
Accurate diagnosis of certain problems may require other tests, including the sam-
pling of water or media. Many tests can be performed only by an outside laboratory
or are commonly performed off-site, such as indicator plant series bioassays for
many viruses other than INSV and TSWV.
Tospovirus Test Kits
The regular sampling of symptom-free plants for the presence of virus can be very
beneficial when you are working with propagation material or crops that are highly
susceptible, serve as symptom-free carriers of virus, or tend to develop delayed
symptoms. On-site test kits allow growers to detect and confirm virus presence more
quickly than is possible with outside laboratory tests. For propagators and growers
of sensitive crops that will benefit from frequent testing, the on-site tests can be
more economical than an outside laboratory.
Virus Indicator Plants
Indicator plants are easy to use for detecting the presence of thrips that can transmit
tospoviruses. Although we do not detail the methods here, you can use indicator
plants in combination with test kits. Indicator plants are used because they quickly
develop blackish lesions when an infectious thrips feeds on their leaves, so they
reveal when and where infectious thrips occur in growing areas. The petunias
‘Burgundy Madness’ and ‘Blue Carpet’ are good indicator plants for INSV and TSWV
according to University of California research. Dark lesions develop on leaves of
these petunias within about 2 to 4 days after feeding by infectious thrips. Indicator
plants that detect virus presence in thrips are distinct from the test kits or the pre-
viously mentioned plant indicator series used to identify the virus species in crop
plants. Indicator plants and test kits are complementary tools that can be used
together in an overall virus monitoring program.
Root Decay Test Kits
Kits for detecting fungi were not developed and optimized for presymptomatic
detection of disease. Exercise caution if you use test kits for routine monitoring
to help you to decide whether or not root decay pathogens are developing, even
though symptoms are not apparent. Since all Phytophthora species are plant
pathogens, the regular sampling and testing of root tips in the bottom 1 or 2 inches
of the root ball of container plants may work well for Phytophthora. Many Pythium
and Rhizoctonia species are weak pathogens or are nonpathogenic, however. These
non-pathogenic fungi commonly colonize very fine roots or dead roots killed by
improper irrigation, solar heating of containers, or other causes. Before you base
your root decay treatment decisions upon the results of tests on asymptomatic
plants, draw on other information collected in an overall disease monitoring program.
EASY ON-SITE TESTS FOR FUNGI AND VIRUSES IN NURSERIES AND GREENHOUSES 5
Besides their use confirming the presence of infected plant tissue and possible
routine monitoring use in crops, ELISA tests can detect root decay fungi in water
and soil. Water tests can be especially important when you are using surface or recy-
cled water for irrigation. Water or soil testing may require special sampling tech-
niques; for more information, contact your Cooperative Extension advisor or agri-
cultural product supplier.
ESTABL ISH ING AN OVERALL D ISEASE MONITORING PRO GRAM
You can use test kits in combination with other information to make good pest man-
agement decisions. Learn which pathogens can attack the crops you are growing.
Scout the crops regularly (at least weekly) for signs and symptoms of disease. Each
time that you monitor for root disease, select at least a few plants from different areas
of each crop and remove the plants from their containers or gently scrape or wash
away the soil. Examine the roots and crowns for browning, softness, or other early
indications of disease. Also inspect aboveground parts for discoloration or wilting
and fungal growths, which may indicate more advanced stages of disease.
Understand the conditions and practices that promote diseases and regularly
scout for and remedy disease-promoting conditions and practices. Poor sanitation,
inadequate drainage, and improper irrigation are primary conditions promoting root
decay. Where virus is a concern, use yellow sticky traps (or possibly blue traps for
thrips) throughout growing areas to provide early detection of insect vectors.
Routinely scout growing areas and remove alternate hosts and reservoirs of disease
and insect vectors, including weeds, crop residue, and old plants that will not
be marketed.
S U G G E S T E D R E A D I N G
Ali-Shtayeh, M. S., J. D. MacDonald, and J. Kabashima. 1991. A method for using
commercial ELISA tests to detect zoospores of Phytophthora and Pythium species
in irrigation water. Plant Disease 75: 305–311.
MacDonald, J. D., J. Stites, and J. Kabashima. 1990. Comparison of serological and
culture plate methods for detecting species of Phytophthora, Pythium, and
Rhizoctonia in ornamental plants. Plant Disease 74: 655–659.
Xia, J. Q., C. L. Sutula, and D. B. Marti. 1996. Development of a greenhouse test for
tomato spotted wilt virus and impatiens necrotic spot virus. Acta Horticulturae
431: 193–198.
EASY ON-SITE TESTS FOR FUNGI AND VIRUSES IN NURSERIES AND GREENHOUSES 6
S O M E S U P P L I E R S A N D D I S T R I B U T O R S
Agdia, Inc.
30380 County Road 6
Elkhart, IN 46514
phone: 1-800-622-4342 or (219) 264-2014
World Wide Web: http://www.agdia.com
Gempler’s, Inc.
211 Blue Mounds Road
P.O. Box 270
Mt. Horeb, WI 53572
phone: 1-800-382-8473 or (608) 437-4883
World Wide Web: http://www.gemplers.com
Neogen Corporation
620 Lesher Place
Lansing, MI 48912
phone: 1-800-234-5333 or (517) 372-9200
World Wide Web: http://www.neogen.com
U C I P M W O R L D
