Agriculture Reference
In-Depth Information
Incubation
Incubation tests require that seeds be subject to conditions that select for and optimize growth of the target
pathogen on either moist blotters or culture plates (Figs. 10.6 and 10.7). Assays usually require pre-treat-
ment with a chemical to disinfect the seed surface to remove microbial contaminants. Seeds are then incu-
bated under precisely deined environmental conditions such as temperature, humidity, light, and period of
incubation. In the case of culture plate tests, the medium used is also speciied for the target pathogen. After
incubation, fungal pathogens are identiied by colony characteristics including color, texture, fruiting body
production, and rate of growth. Diagnosis is also conirmed by microscopic examination of fruiting body
type, size, or structure.
Since the early 1980s, major advances in the development of selective media for different groups of
fungal and bacterial pathogens have greatly increased the utility of culture plate testing, particularly for bac-
terial assays. Bacterial assays are usually carried out in three steps: extraction, isolation, and identiication.
The extraction phase normally requires placing whole or ground seed samples in an aqueous solution for a
speciied time period at a deined temperature. Bacteria are released from the seed tissues into the solution.
Aliquots of the extract solution are then cultured in a dilution series on a medium selective for the target
pathogen. Putative colonies of the pathogen may undergo sub-culturing to remove microbial contaminants.
Pure cultures are then subjected to biochemical, serological, or DNA tests to conirm the identity of the
target bacterium. Cultures also may be inoculated onto seedlings to conirm their pathogenicity. Figure 10.8
illustrates this three step process for the
Pseudomonas syringae pv glycinea,
the cause of bacterial blight of
soybean.
Incubation tests are capable of detecting a broad range of fungal and bacterial seedborne pathogens.
The sensitivity of these tests also may be high, particularly if a selective medium is used. Sensitivity may,
however, be reduced if seeds are heavily contaminated by saprophytes. By manipulation of seed pretreat-
ment, incubation tests may be used to quantify the component of a pathogen that is both internal and/or
external to the seed coat. A limitation of incubation tests is that most protocols require incubation times
of about 7 days. This can create problems with space and time in high-volume testing laboratories. A well
equipped plant pathology laboratory is also needed to carry out incubation tests, although it is possible to
carry out blotter testing under more rudimentary conditions.
Grow-out Tests
In this procedure, seeds are planted in the ield or greenhouse in the absence of other inoculum sources
such as infected crop residues or insect vectors. After a deined period of growth, under environmental
conditions favorable for transmission of the pathogen from seed to seedling, seedlings are inspected for
symptoms produced by the seedborne pathogen (Fig. 10.9).
Because the seedborne pathogen must be transmitted to the seedling and express pathogenicity before
it can be detected, grow-out tests have an advantage over other protocols in giving a more accurate estimate
of the risk of transmission of the pathogen to the new crop. However, sensitivity can be low in grow-out
procedures, making them unsuitable for methods that require estimates of the incidence of seedborne infec-
tion, regardless of transmission potential. The test also has signiicant limitations in that it can only be
applied when plant symptoms are distinct enough to provide an accurate identiication of the target patho-
gen. Grow- out tests also require much time, space, and labor.
