Koch's postulates in plant pathology
Four steps that prove a microbe causes a plant disease, lab methods, exceptions for viruses and obligate parasites, and molecular forms.
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A diseased plant is usually covered in microbes. Some are the cause of the disease, many are harmless passengers that arrived after the tissue started to die, and a few are beneficial. Before a disease can be controlled, a new pathogen reported, or a quarantine imposed, plant pathologists must prove which organism is responsible. The classic standard of proof is Koch's postulates.
Koch's postulates are among the most frequently asked questions in plant pathology exams, and their practical application is a core laboratory skill.
Where the postulates came from
The idea that microbes cause plant disease came before the postulates. In 1807, Bénédict Prévost showed that spores of the bunt fungus caused bunt of wheat and that copper sulphate prevented it. In 1861, Anton de Bary proved that the oomycete now called Phytophthora infestans caused potato late blight, the disease behind the Irish famine. In 1878, T. J. Burrill showed that fire blight of pear and apple was caused by a bacterium, the first bacterial plant disease proved.
Robert Koch, working on anthrax (1876) and tuberculosis (1882) in animals and humans, developed pure-culture methods that made rigorous proof possible. The rules were formally stated by his colleague Friedrich Loeffler in 1884 and have been applied to plant diseases ever since.
The four postulates
- Association: the suspected organism must be found in all plants with the disease.
- Isolation: it must be isolated and grown in pure culture, and its characteristics described.
- Inoculation: the pure culture, inoculated into healthy plants of the same species or variety, must produce the same disease.
- Re-isolation: the same organism must be recovered from the inoculated plants and shown to match the original.
Doing it in the laboratory
Isolation
- Choose fresh tissue from the advancing edge of a lesion, where the pathogen is active and secondary organisms are fewer.
- Surface-sterilise small pieces, typically in about 1 per cent sodium hypochlorite for one to two minutes, sometimes after a quick dip in 70 per cent ethanol, then rinse in sterile water.
- Plate onto suitable media: potato dextrose agar for many fungi, nutrient agar or King's B for bacteria, and selective media for particular groups.
- For bacteria, a streak or ooze test (cut stems in water releasing milky streams of bacteria) helps.
- Purify by single-spore or hyphal-tip isolation for fungi and single colonies for bacteria.
Identification
Morphology of spores and colonies, biochemical tests, and increasingly DNA sequencing of standard barcode regions, such as the ITS region for fungi and 16S rRNA or multilocus sequences for bacteria.
Inoculation
The method should mimic natural infection: spraying spore suspensions, wound inoculation, root dipping, soil infestation, or stem injection. Plants are kept in conditions favourable to disease, often high humidity, and compared with mock-inoculated controls treated with sterile water or medium.
Proving the cause of a wilt
A grower reports wilting tomatoes with brown discoloration inside the lower stem.
Association and isolation. Stem pieces from 50 wilted plants are surface-sterilised and plated. A fungus with pink-purple colonies and sickle-shaped spores, identified as Fusarium oxysporum, grows from 42 of them.
- Isolation frequency = 42 ÷ 50 × 100 = 84 per cent. Other fungi appear only occasionally.
Inoculation. Roots of 20 healthy seedlings are dipped in a spore suspension of a pure culture (about 106 spores per millilitre) and transplanted; 20 control seedlings are dipped in sterile water.
- After three weeks, 18 of 20 inoculated plants wilt (90 per cent), with the same internal browning; none of the 20 controls do.
Re-isolation. The same fungus is recovered from 17 of the 18 wilted plants (94 per cent), and its DNA sequence matches the original isolate.
All four postulates are satisfied: this isolate causes the wilt. Testing it on varieties with known resistance genes could then identify its race, which decides which resistant varieties will work.
When the postulates cannot be applied directly
- Obligate parasites, such as rust fungi, powdery and downy mildews, cannot be grown on artificial media. They are maintained on living host plants, and spores from a single pustule serve as the "pure culture".
- Viruses and viroids cannot be cultured. In 1937, T. M. Rivers proposed modified postulates for viruses. Proof relies on transmission from purified virus or infectious clones, electron microscopy, serology and sequencing.
- Phytoplasmas and some fastidious bacteria, such as the bacterium associated with citrus greening, have not been grown reliably in pure culture, so their causal role rests on consistent association, transmission by grafting or insect vectors, and molecular evidence.
- Disease complexes, where two organisms together cause disease (such as root-knot nematodes and Fusarium wilt), need inoculation with both.
- Latent infections and endophytes: some organisms live in healthy plants without symptoms, so finding them in diseased plants is not enough.
Molecular Koch's postulates
In 1988, S. Falkow proposed molecular Koch's postulates to show that a specific gene is responsible for virulence: the gene is found in pathogenic strains, knocking it out reduces virulence, and restoring it restores virulence. In 1996, D. Fredricks and D. Relman proposed sequence-based criteria for linking microbial DNA to disease when the organism cannot be cultured. Plant pathologists also use infectious clones: a DNA copy of a virus genome that, introduced into a plant, reproduces the disease.
Key points
- Koch's postulates: associate, isolate in pure culture, inoculate to reproduce the disease, and re-isolate the same organism.
- Loeffler stated them in 1884; Prévost (1807), de Bary (1861) and Burrill (1878) had earlier shown microbes cause plant diseases.
- Isolate from lesion margins after surface sterilisation; purify by single-spore or single-colony isolation; include healthy controls.
- Obligate parasites, viruses, phytoplasmas and fastidious bacteria need modified approaches.
- Molecular Koch's postulates link virulence to specific genes.
What the exams ask
- School-leaving and entrance papers: stating Koch's postulates, and the history of plant pathology.
- Degree and research-fellowship entrances: isolation and purification techniques, inoculation methods, limits for obligate parasites and viruses, Rivers' postulates, and molecular postulates.
- Professional and quarantine examinations: diagnosing new diseases, confirming pathogen identity, and reporting new records.
Described in general terms: the kind of examination, not a past paper. Laisha never reproduces exam questions.
Questions people ask
Why isolate from the edge of a lesion?
The centre of an old lesion is dead tissue, often colonised by secondary microbes. The advancing margin is where the pathogen is actively invading, so it gives the best chance of isolating the cause.
What if inoculation produces milder symptoms than in the field?
Conditions in the experiment may differ from the field, or the isolate may have lost aggressiveness in culture. Repeating inoculations under more favourable conditions, or with fresh isolates, usually resolves it.
Are Koch's postulates still needed with DNA tests?
DNA tests detect organisms quickly, but presence is not proof of cause. For a new disease or a new host, fulfilling the postulates, or their molecular equivalents, remains the standard of proof.
Sources
- Agrios, G. N. Plant Pathology, 5th edition. Elsevier Academic Press, 2005. Chapter 1.
- Schumann, G. L. and D'Arcy, C. J. Essential Plant Pathology, 2nd edition. APS Press, 2010.
- Falkow, S. "Molecular Koch's postulates applied to microbial pathogenicity." Reviews of Infectious Diseases 10, S274–S276 (1988).
- Fredricks, D. N. and Relman, D. A. "Sequence-based identification of microbial pathogens: a reconsideration of Koch's postulates." Clinical Microbiology Reviews 9, 18–33 (1996).
- Rivers, T. M. "Viruses and Koch's postulates." Journal of Bacteriology 33, 1–12 (1937).
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