Testing Seeds for Seedborne Organisms

ALICE M. ANDERSEN AND CHARLES M. LEACH.

THE TESTING of seeds for purity and germination is a recognized practice in many countries. In recent years there has come about a growing realization among scientists, seedsmen, and seedgrowers that testing for purity and germination is not enough and that international methods for testing the most serious seed-transmitted organisms are needed.

C. R. Orton, of the West Virginia Agricultural Experiment Station, in 1931 estimated losses in the United States caused by seedborne diseases to amount to 2.3 percent of the total crop of wheat, 3.1 percent of the barley crop, 3.2 percent of the oats crop, and 6.1 percent of the bean crop. The losses may appear insignificant for the country, but they are significant losses to the growers and to the sections where the losses occur.

A list of seedborne diseases compiled by Mary Noble of Scotland, J. de Tempe of Holland, and Paul Neergaard of Denmark, under the auspices of the International Seed Testing Association and published in 1958 by the Commonwealth Mycological Institute in London, registered approximately 900 plant diseases that may be spread by seedborne organisms.

Four groups of organisms commonly associated with seed cause plant diseases. In order of importance they are the fungi, bacteria, viruses, and nematodes. Some other diseases of seeds result from deficiencies of plant nutrients and from undetermined causes.

Seedborne pathogens affect directly and indirectly the quality of seeds in commerce. Of primary importance is the fact that seedborne plant pathogens introduce diseases such as smuts into newly sown crops thereby causing a reduction in yields and quality. The field germination of seed infected with seedborne pathogens may be reduced because the pathogens attack and kill the seedlings.

Similarly, the accuracy of germination tests in laboratories may be affected by certain seedborne pathogens, which in some instances cause a browning of the seedlings or attack and kill young seedlings during the tests. The viability of seeds in storage may be reduced by molds, which are unimportant in the field but may attack the dormant stored seeds.

Many kinds of crop seeds are treated with fungicides to eradicate seedborne pathogens and to protect them from pathogenic soilborne organisms, but not all seedborne pathogens would be killed even if we treated all seeds.

If buyers of seeds are to be assured of purchasing seeds free from harmful pathogens, some means is needed whereby the amount of infested seed within a seed lot can be ascertained.

Methods for detecting many seed-borne pathogens have been devised. Some pathogens, however, are not easily detected when seedborne, and field inspection of the growing seed crop is probably more practical in those instances than attempts to detect them within seed lots.

A commercial seed lot must be sampled before it can be tested for the presence of pathogens. We have little information on the precise methods for sampling seeds for the presence of pathogens or the size of the samples to be used for pathological testing. The dividers commonly used in North American and European seed-testing laboratories are not entirely suited for the mixing of seeds for pathological testing, principally because they cannot be easily cleaned and disinfected between samples. We believe the Patterson-Kelley "p K" type of mixer overcomes this objection.

DETECTING seedborne fungi on seeds is accomplished by a number of methods. Examination of dry seeds with the naked eye and at magnifications of 10 to 30 times reveals a number of plant pathogens that occur mixed with the seeds as fungus bodies (for example, sclerotic) or have converted the seed into fungus structures (e.g., ergots). Sclerotic of the fungi Sclerotinia and Typhula may be mixed with seeds of clovers, crucifers, grasses, and other crop seeds. The fungus Claviceps Purpurea, the cause of ergot of grasses, often is mixed with seed as ergots.

Also detected by visual examination are certain fungi that form small fungus structures on or in the seedcoat.

Septoria apii, which causes a blight of celery, forms small structures (pycnidia) embedded in the seedcoat. It can be detected at low magnifications. The fungus Septoria macropoda, a pathogen of grasses, forms pycnidia on the plumes of bluegrass seed.

Other fungi may cause such symptoms as sunken, discolored, and dead areas on the seeds. Occasionally the presence of these symptoms may be used to detect pathogenic organisms. Identification of pathogens on the basis of disease symptoms alone is generally not recommended, for errors are easily made.

WHEN SEEDBORNE fungi are present as microscopic spores on the surface of seeds, it is almost impossible to detect them by visual examination at low magnifications. The spores have to be removed from the seed by washing and then examined with a compound microscope. Several procedures have been used.

For the covered smut diseases of cereals, a known weight of seed is added to a measured volume of water to which a little detergent has been added. The seeds are then shaken, and the washings are removed by decanting. The spores in suspension are allowed to settle at their own speed or more rapidly by the use of a centrifuge. The number of spores for a known amount of seed (spore load) is determined microscopically with a hemocytometer counting chamber.

The spores of some pathogens may be associated in such great numbers with individual seeds that merely mounting the seeds in drops of water, tearing the seeds apart, and then examining the exuding spores microscopically is sufficient for detection. This procedure has been used for detecting the honey dew stage of Claviceps Purpurea (ergot of grasses) and blind seed fungus of ryegrass (Gloeotinia temulenta).

In Cereal Chemistry, volume 37, 1960, Robert M. Johnson described five techniques for the determination of covered smut in wheat light transmittance, sedimentation, catalase activity, light reflection, and light absorption. The first four techniques measure the external smuts. The last employs a smut meter, which measures the internal and external smuts that contaminate the seeds.

THE TECHNIQUE of placing disinfected or untreated seeds on moist blotters and incubating them at specified temperatures has been used in Europe for detecting a number of seedborne pathogens, such as species of Helminthosporium and Fusarium. After incubating the seeds for a prescribed period, the pathogens are identified on the basis of disease symptoms of the seedlings or microscopic examination of individual seeds for the presence of fruiting structures of fungi.

Disease symptoms should be used with caution for identifying seedborne pathogens because different pathogens can cause similar symptoms of disease. If identification is made on the basis of microscopic examination of fruiting structures, each seed must be examined individually at relatively high magnifications. This is tedious and slow if samples of 400 seeds or more are used.

A modification of the blotter method has been devised, in which a low concentration of 2,4-D is added to the water used for moistening the blotters.

The 2,4-D inhibits germination of the seeds and facilitates microscopic examination of the seeds because they all remain in one plane, unlike the germinating seeds, which lie in many planes.

When the blotter method is used to obtain reproducible results, the incubation temperatures, length of incubation, and type and intensity of lighting must be standardized.

THE AGAR plating method is used widely for detecting fungus pathogens. Surface disinfected or untreated seeds are placed on a variety of agar media. The pathogens grow from the seeds and are identified by their macroscopic colony characteristics. When there is any doubt about their identity, microscopic examinations are made. Malt extract agar and potato dextrose agar are used oftenest.

When the media plating method is employed, standardization of temperatures, length of incubation, pH of the media, and light are important if reproducible results are to be obtained.

Research by Charles M. Leach at the Oregon Agricultural Experiment Station has shown that continuous irradiation with near ultraviolet (UV) 3,200A-4,000A from fluorescent black lamps and light from commercial fluorescent lamps greatly aids sporulation and subsequently the identification of a number of seedborne fungi. Other species of fungi (for example, Helminthosporium um oryzae) are induced to form spores only if periods of near UV are alternated with dark periods.

Clyde M. Christensen, in Botanical Review, volume 23, 1956, described procedures for detecting molds that cause deterioration of cereal seed in storage. Seeds are pulverized in a medium of sodium chloride and agar. Then the suspension is plated in salt malt agar. Storage molds, particularly species of Aspergillus and Penicillium, grow well on this medium, but the growth of the saprophytic fungi commonly found on cereal seeds is retarded.

By growing seeds in soil, sand, and ground brick in greenhouse and laboratory, one can detect certain fungus pathogens that sometimes are difficult to detect by other methods. Identification usually is based on disease symptoms. Symptoms of loose smut of oats can be detected within 2 months when seeds are sown in the greenhouse in dry, cool soil. A combination of laboratory and greenhouse methods has been used to detect the fungus Helminthosporium gramineum, which causes barley stripe.

The embryo method was developed for determining loose smut of barley and wheat. The seeds are softened with sodium hydroxide to remove the embryos. The embryos are cleared in chloral hydrate or lactophenol and examined microscopically for the presence of fungus hyphae. The hyphae are more clearly seen by staining with cotton blue.

SEEDS infected with bacterial pathogens generally are not distinguishable from healthy seeds. To detect seeds infected with bacteria, two main methods are available.

The first is to grow plants in the laboratory or greenhouse under optimum conditions for the development of the pathogen until disease symptoms are evident. This method has been used in England for the detection of halo blight disease of beans.

A second method is the rapid phage-plaque count technique. It employs bacteriophages (bacteria-destroying agents) capable of lysing, or dissolving, specific bacterial pathogens.

Bacteriophages have been developed for Pseudomonas pisi, a bacterial pathogen of peas; P. atrofaciens of wheat; P. coronafaciens of oats; Xanthomonas translucens f. spp. hordei, secalis, and hordeiavenae; Corynebacterium insidiosum of alfalfa; and other seedborne bacterial plant pathogens.