1 Koch’s Postulate for Biotroph and Necrotroph Pathogens

Transcript

Hello! I welcome you to the second week of the course Detection, Diagnosis and Management of Plant diseases. In the first talk of this week we’ll be talking about Koch postulates for biotrophic and Necrotrophic pathogens. We all know that Koch’s postulates has been given by Robert Koch while he was working with the etiology of cholera and to ecologies but the postulates he developed for establishment of the microorganism associated with cholera and tuberculosis convey extended for other plant pathogens which are microbial in nature. So, there are four steps basically in Koch postulates. In the first step there is a must association of microorganisms with all individual plants affected by the disease but those microorganisms should not be associated with healthy plants. In the second step the microorganisms should be isolated from the disease plant and grown in culture. This is very important and the third step is when introduced into a healthy plant the cultured microorganism should cause disease. And the fourth postulate is the microorganism must then be re-isolated from the experimental host and found to be identical to the original microorganism. So in the four steps it is essential to carry out for most of the microbial pathogens which are associated with plant diseases. But sometimes there are certain pathogenic causes or the host properties that is a source difficulties in establishing the cause postulates and that is why the disease should be produced in a new host but it is not a must, because there may be certain asymptomatic carriers, or the host immunity factor, or the genetic resistance, that are possible and these factors may not allow development of the disease even though the pathogenic micro is inoculated to an experimental host. So Koch postulates for biotrophs can be different from Koch postulates from microtrophs, particularly in the steps of inoculation and isolation of the pathogen.

So Koch postulates for stem rust of wheat can be taken an example for biotrophs where, wheat seeds are of a susceptible cultivars sown in the pots and the seedlings are grown until the first leaves are fully emerged. Then during inoculation leaves should be rubbed gently between the moistened fingers so as to remove the waxy layer of the surface of the leaves which hinders penetration of germ tube of the pathogen spores. Then spores should be suspended in distilled water and with a drop of Tween 20 and the spore suspension should be spread until runoff with an atomizer. The seedlings of inoculated and incubated a relative humidity of about 100 percent and the temperature should be of around 22 degree Celsius in a plastic chamber for at least 24 hours.

This is important for giving the proper facility for development of the spore germination, germ tube development and penetration . By the rust pathogens transfer of seedlings to a greenhouse bench should be done and the temperature should be maintained at around 18 to 27degree celsius and at least it should be kept for two weeks for development of the symptoms. Samples with viable spores be selected to multiply in a columns for generation of monopustule isolates. So the inoculation with monopustule isolates is important because if it is not monopustule then there may be a mixture of races that may be present in the same leaves and it may not lead to a differentiating response in the host plants. So monopustule isolation before sporulation should be done for spore suspension preparation in case of wheat rust pathogen so that a clear-cut distinct Koch postulate could be proved. Then the question arises how to select a monopustule isolate for identification of races. The leaves with monopustule all infections should be identified prior to sporulation and then isolated because, once the sporulation takes place then there may be a mixture of spores that may be present in the suspension. In case where the pustules are aggregated and no isolated past will occur, inoculation is repeated on the susceptible cultivar until separate pustules are developed and monopustule isolates are generated. As long as we get monopustules we can keep on repeating spraying of the monopustule on wheat cultivars and a susceptible cultivar so that we are able to generate a monopustule and develop spore suspension from the same single monopustule for identification of wheat rust pathogen races. The generated monopustule isolates are then further multiplied on a susceptible host until sufficient urediospores are collected for differential host test. So this is very important to generate monopustule for differentiation of a race of wheat rust pathogen. Then the cost postulates for necrotrophs may be slightly different from biotrophs. Let us take the example of Alternaria species where Foliar spray of the spores is also done but after culturing of the pathogen on an agar plate. So in case of Alternaria the pathogen should be isolated and cultured on agar medium at the very beginning. Then once the sporulation takes place there distilled water should be placed on the surface of the plate and then slightly vortex thing should be one. After that the spore suspension should be decanted on a beaker and then it should be collected in a hand atomizer and then healthy plants are sprayed till water drips off from the leaves. Then the leaves are covered with a plastic bags for at least 24 hours and allow the plants to grow in a glass house /net house till the symptoms are developed. So in this way the Koch postulates are also proven in necrotrophic pathogen but isolation of the pathogen as well as handling of spore suspension from different sources matters.

Similarly, another Necrotrophies the soil borne pathogen that is Fusarium. Here the like panacotic pathogen is not a Is not leave inoculated rather it is soil drenched. Spores of the Fusarium were collected in a similar way just in the case of all Alternaria species but after collection in the beaker the 30 to 50 ml of the spore suspension is then drenched in the pot around the plants and the pots are kept for appropriate temperature for development of the disease and development of the wilt Symptoms. So in case of Fusarium we have seen here that instead of spraying of Spore suspension the pathogen is soil drenched around the plant in the pot Soil. Similarly, in case of collar of pathogen that produces mycelia and sclerotia by Sclerotium rolfsi, the application method is inoculation of Mycelia and sclerotia directly into the soil. The pathogen is isolated from an infected plant part it may be a stem or colaor and obtain pure culture on a suitable agar medium. Then inoculate the fresh mycelium disc on cereal grains priorly autoclaved and packed in a polythene bag. Allow the pathogen mycelium to grow and develop sclerotia. Then take out cereal grains colonized by the Sclerotium rolfsi pathogen mycelium as well as sclerotia of about 5 to 10 grams and the care should be taken that adequate soil moisture is already prevailed and the 5 to 10 gram grains should be inoculated into topsoil in pots and mix with the topsoil Thoroughly. Then allow the plants to grow till symptoms are developed. So again this is another necrotrophic pathogen but the method of inoculation for testing Koch postulate is different. Here the pathogen is cultured on cereal grains and the colonized grains with the pathogen mycelium and sclerotia is then inoculated into topsoil of the plants and then allowed to develop infection and symptom in the pot till the appropriate symptoms are observed. So here we have seen that for different pathogens the proving of Koch postulates may have some differential steps to be taken care of so that appropriate disease symptom is generated in the host plants to prove the pathogen to be associated with the disease-causing agent. So with this we come to an end of today’s talk and in the next talk we will talk about the concepts of pure culture and selective media and, we will see their how pure cultures of fungal and bacteria is done and what is the role of selective media in obtaining the pure culture of the plant pathogens.

Thank you very much.