Submission Guidelines

The Plant and Pest Diagnostic Clinic (PPDC) diagnoses diseases of all types of plants. If insects or cultural problems are found, these are also investigated. To submit, obtain and complete the Plant Diagnostic Form from the PPDC Homepage or from your county extension office. Select as much recently affected plant tissue as possible, making an effort to represent all stages and aspects of the problem. Provide material which shows a range of symptoms present on the plant. Dead tissues are often useless for diagnosis, but some dead material can be included to represent the range of symptoms. Do not wash samples, as this may remove pathogen structures and encourage growth of secondary organisms. Several plants of the same kind, with the same symptoms, may constitute a representative sample and be covered by one submission fee.

Including photographs or digital images of the affected plant(s) in their environment is encouraged, but close- up photos usually aren’t necessary. Enclose plant diagnostic samples in sturdy plastic bags to prevent tissue desiccation. Never add water or wet paper towels, as this encourages growth of secondary microorganisms and may lead to sample decay. Avoid sending wet or dirty leaves for the same reasons.

If plants are potted, submit entire plants in their pots and package well to prevent toppling during shipment, which can result in rotting of above-ground tissue due to soil contamination. When roots and soil are included from field-grown plants, enclose the roots in a plastic bag and tie off at the stem, or pack roots in a separate plastic bag.

It is very important to submit samples soon after collection. If submission must be delayed, keep samples cold, but not frozen, before mailing. Be sure to provide dates of sample collection and mailing. This information, along with a complete description of the problem, is needed so that damage or contamination of the sample during transit will not be confused with the real problem.

Consider that the source of the problem may be abiotic, or non-living. Because of this, be sure to complete the form, giving information on plant care, fertilizers, specific pesticides and other chemicals that have been used on or in the vicinity of the plant. Also, be assured that if the sample is deemed insufficient by diagnosticians, one free resubmission will be granted. Only diagnosticians can make this determination.

Packaging and Mailing Samples

It is best to pack samples in boxes to prevent crushing. Use ample packing material to prevent shifting and tumbling in transit. This will help to maintain sample quality during shipment. Consider the nature of the sample before mailing, as samples delayed in the mail are more apt to deteriorate in transit. Mail samples early in the week and they should arrive during the same week. The Clinic supplies plastic bags and insect vials for the counties to use for sending samples. County offices can request these by sending a form from the website to the Lab Coordinator, Diana Low, who will package and mail them as soon as possible after receipt of the request.

Replies

Depending on sample load at the time, replies can be expected from 7 - 10 days following sample receipt in the Clinic. During cooler seasons, replies may come more quickly. All replies are sent via email to clients and also to the affiliated county extension office. Agents included on the form will also receive a copy. If the client has no email address, even though preliminary reports will generate, only the final report will be mailed

Leaf spots:

Collect at least 6 to 12 leaves representing all stages of infection. For plants with small leaves, cut off a branch with leaves intact. Highly succulent leaves should be treated as described in the next section.

Fruits or other fleshy tissues:

Avoid packing fruit, fleshy leaves or other fleshy organs showing advanced stages of rot; select early stages of infection or damage. Wipe off excess moisture and wrap fruits individually in dry paper and place in a paper bag. Such samples are highly perishable so overnight mail is recommended.

Stem lesions, diebacks, cankers, and galls:

Select branches with active lesions or young galls. Cut branches to include the affected area and a healthy portion on the same branch, if possible. Completely dead branches and twigs are generally undesirable for diagnosis, but may be included, along with more recently declining ones, to show a range of symptoms.

Turfgrass samples:

Cut out blocks of sod (grass with roots and soil attached) of at least 6 x 6 inches, to include the margin between affected and healthy areas. Place in a sturdy plastic bag and press to release trapped air.

Plants exhibiting wilting, yellowing, and general decline:

Submit entire plants, if possible. If they are container grown, submit several affected plants in their pots, if possible. If they are field grown, carefully dig them up and place soil-covered roots in one plastic bag, tie at the trunk, and secure another bag over the top. For large trees and shrubs, submit branches or other parts showing typical symptoms in a plastic bag. Include a handful of fine feeder roots, plus a few larger ones, of at least ¼ inch in diameter. Place roots in a separate plastic bag with a small amount of moist soil. Make sure that the roots submitted are from the plant itself, not from weeds, grass or ground covers growing below the affected plant.

Viral Symptoms:

Mosaic patterns, chlorotic ring spots, and distorted new growth are common symptoms of virus diseases. Only a select few viruses can be identified through the PPDC but samples can be sent from the Clinic to a commercial lab at an additional cost, if desired. Send virus assay samples by overnight delivery.

Nematodes:

If nematode problems are suspected, soil from the sample can often be used to test for these microscopic round worms, but an additional fee will be required. If the Diagnostician suspects nematodes, clients will be notified and asked about testing. If agent suspects nematodes, clients should be encouraged to submit nematode assay samples. Collect a pint of soil from the advancing margin of decline and enclose in a nematode bag or small plastic bag. Do not allow the soil to dry out or become overheated during shipment. It is helpful when clinic and nematode samples are submitted separately, that this information be provided on the diagnostic sample submission form. If more than one sample is submitted, be sure to label the bags and provide these field ids on the form as well. Nematode assay results will be sent by NAL lab personnel.

Nutritional Analysis:

Soil or tissue analysis samples should be sent directly to the Agricultural Service Lab (ASL). Soil or tissue analysis samples sent with PPDC samples will be delivered to the ASL, but there will be a delay in submission and results. A completed form and a separate payment must be included for these additional tests. Collect a pint of soil at a uniform depth for pH and nutrient analysis of mineral soils. For soilless mix, tissue nutrient analysis or other ASL tests, consult their website or ASL personnel (864-656-2068) for more information.

Aquatic plants consist of vascular plants and algae. Often, they are entangled in a mass, making it difficult to separate one from the other. However, it is important to attempt to do this, as each type of plant should be submitted separately, and a fee paid for each type. Try to assess which type is most prevalent in the pond and attempt to collect that one as the sample. As with terrestrial plants, submitting vascular aquatics with flowers, fruits or seeds is preferred for the most accurate identification.

Place aquatic plant samples in tightly sealed plastic jars or double bag them in zip-lock bags that are taped closed. Samples of algae should be submitted in water in plastic bottles. Glass jars are discouraged, as these add to shipping costs and are more vulnerable to breakage if not packed properly.

AFB is a regulated bacterial brood disease that results from the infection of honey bee larvae with Paenibacillus larvae. It is the most destructive of the honey bee brood. While it only attacks larvae, AFB weakens the colony and can quickly lead to death in only three weeks. AFB is most commonly transmitted through spores of the bacterium, which can be dormant in the colonies or contaminated equipment for 70 years or more. When nurse bees feed larvae with food contaminated with spores, the spores turn into a vegetative stage that replicates in the larval tissue leading to its death. Larvae killed by these bacteria have a unique "foul" odor that gives this brood disease its name. New spores form after the larvae die. Each dead larva can contain as many as 100 million spores. Death typically occurs after the cell is capped, during the last two days of the larval stage or first two days of the pupal stage. When the larva remains, and bacterial spores dry out, they form a scale that is glued to the cell. This is typical of AFB and can be used to diagnose this disease. There is no cure for AFB; beekeepers must act when the disease is found.

EFB is a bacterial disease that affects honey bee larvae before the capped stage. It is characterized by dying and dead larvae which can appear curled upwards, brown or yellow, melted, and/or dried out and rubbery. The causative bacterium, Melissococcus plutonius, is ingested by honey bee larvae after which the bacterium competes for food inside the larvae. If the bacterium outcompetes the larvae, the larvae will die before the cells are capped. Alternatively, the bees may survive until adulthood if the larvae have sufficient food resources. EFB should not be confused with AFB, which is caused by a different bacterium and has different symptoms and control requirements. EFB is more problematic in situations where forage nectar is sporadic, or other situations that result in fewer nurse bees in colonies to feed larvae. At the onset of nectar flow in early spring, forage recruitment of house bees may increase rapidly, resulting in few bees in colonies to feed honey bee larvae. Often, when the nurse bee to larvae ratio stabilizes later in the season, or remains stable throughout a season, symptoms disappear. However, this disease can occur throughout a season and will sometimes not clear up on its own. In severe cases, colony death can occur. Also, yearly reoccurrence of EFB from contaminated combs and equipment can occur. The bacterium that causes EFB does not produce spores, but contaminated combs can still reinfect honey bees in subsequent years.

Materials Needed:

• Cotton swabs (Q-tips)
• Matchstick or wooden coffee stick
• Aluminum foil
• Ziploc bag
• Sample submission form (Bee Testing Form)
• Envelope for shipping

Guidelines:

• Select the frame from which the sample will be collected.
• Use the matchstick or wooden coffee stick to perform a ropy test and mark the cell(s) for sample collection.
• Select the cell(s) from which the smear will be collected.
• Stick the Q-tip in the cell and turn it. This will collect the brood sample.
• Place the Q-tip in the Ziploc bag. Be sure to write the sample identification on the bag.
• Please collect only one sample per colony. If multiple colonies need to be tested, then multiple samples are
  required.
• Complete a sample submission form, include all information.
• Ship the sample, sample submission form, and payment of test fee to the laboratory at:
  Clemson University, MPPD Lab, 511 Westinghouse Rd., Pendleton, SC 29670
• Samples must be kept cool and dispatched to the laboratory on the day they are collected, or frozen as soon
  as possible after collecting (preferably within 12 hours). Otherwise, non-AFB bacteria may grow in the
  sample and interfere with the laboratory test.
• Do not send samples to the laboratory on a Friday or on the day before a public holiday, since the samples
  may grow non-AFB bacteria during the delay before delivery to the laboratory.

Avoid potential stings, and protect yourself and other from serious injuries: wear protective clothing, approach with caution, use a smoker, and know your hive. Obtain professional service as needed.

AHB cannot be distinguished from European honey bees easily, although they are slightly smaller than the European races. A more rigorous identification is achieved by genetic analysis and often is necessary when the suspect bees are a hybrid between the Africanized and European subspecies.

Other differences between Africanized and European bees manifest themselves behaviorally. To the casual bystander, the primary identifying behavioral characteristic of AHB is their heightened defensiveness compared to that of European subspecies.

All honey bees readily defend their nests, and an attack usually means that the victim is too close to the nest. While European races of bees may attack a nest intruder with a few bees (usually no more than 10–20 bees), AHB may attack the same intruder with hundreds of bees. Further, AHB generally defend a larger radius around their nest and require lower levels of stimuli to initiate an attack.

Another behavioral difference between Africanized and European bees concerns colony level reproduction and nest abandonment. AHB swarm and abscond in greater frequencies than their European counterparts. Swarming, bee reproduction at the colony level, occurs when a single colony splits into two colonies, thus helping to ensure survival of the species. European colonies commonly swarm one to three times per year. Africanized colonies may swarm more than 10 times per year. Africanized swarms tend to be smaller than European ones, but the swarming bees are docile in both races. Regardless, Africanized colonies reproduce in greater numbers than European colonies, quickly saturating an area. Further, AHB abscond frequently (completely abandon the nest) during times of dearth or repeated nest disturbance while this behavior is atypical in European bees.

Another common difference between Africanized and European honey bees is their choice of nest locations. AHB are less selective when considering a potential nesting site than are European bees. They will nest in a much smaller volume than European honey bees and have been found in water meter boxes, cement blocks, old tires, house eaves, barbecue grills, cavities in the ground, and hanging exposed from tree limbs, to name just a few places. One rarely finds European colonies in any of these locations because they prefer to nest in larger cavities like those provided by tree hollows, chimneys, etc. As one can imagine, humans inadvertently provide multiple nesting sites for AHB. Therein lies the primary reason AHB are encountered frequently by humans.

A final behavioral curiosity of AHB concerns nest usurpation (or colony takeover) of European colonies. Small Africanized swarms containing a queen often land on the outside infrastructure of a European colony (a wall, beekeeper-managed hive, etc.). As time passes, the worker bees in the Africanized swarm begin to exchange food/pheromones with the European workers from the colony. This gradually ensures the adoption of the AHB into the European colony. Somewhere during this process, the European queen is lost (perhaps killed by AHB as her fate remains uncertain at this point) and the Africanized queen is introduced into the colony, thus becoming the reigning matriarch. European bees do not display this behavior but often fall victim to it, thus creating an Africanized colony from a preexisting European one.

Materials Needed:

• Leakproof container
• Alcohol
• Sample submission form (Bee Testing Form)
• Collection tub
• Scoop
• Funnel
• 10 honey bees

Guidelines:

• Please collect only one sample per colony. If multiple colonies need to be tested, then multiple samples are required.
• Select a frame of honey bees from the colony.
• Make sure the queen is not present on the frame.
• Shake the frame of honey bees into a collection tub, scoop out 10 honey bees, use the funnel to introduce the honey bees into a leakproof container, cover honey bees with alcohol, and secure the lid tightly.
• Complete a sample submission form, include all information.
• Ship the sample, sample submission form, and payment of test fee to the laboratory at:
  Clemson University, MPPD Lab, 511 Westinghouse Rd., Pendleton, SC 29670

Avoid potential stings, and protect yourself and other from serious injuries: wear protective clothing, approach with caution, use a smoker, and know your hive. Obtain professional service as needed.