DE3419382A1 - Method of detecting phytopathogenic microorganisms in the soil and in diseased plants - Google Patents

Abstract

The method involves passing the aqueous phase of a soil sample which has been extracted by shaking through a paper filter. The solution, from which soil particles have been removed, is then sucked through a membrane filter of suitable pore size with the aid of a vacuum filter apparatus. The membrane filter is then cut into strips and at least one strip is used for inoculating a test plant. After the incubation time has elapsed, the test plant is examined for the response of the test plant tissue. The method is less complicated and more reliable. Clear test results can be obtained within a short time.

Description

Method for the detection of phytopathy, elogenic microorganisms in the soil

and in infested plants. The invention relates to a biological one Method for the detection of phytopathogenic microorganisms in the soil and in infested ones Plants (suspected of being infected), especially bacteria of the genera Agrobacterium and Erwinia as well as parasitic slime molds and Chytridiomycetes.

Phytopathogenic microorganisms are common inhabitants of cultivated soils of all continents. They attack their host plants as well as their fruits and cause Tissue destruction or tissue growth.

Plants or products that have been damaged in this way cannot be sold.

Most countries prevent imports and exports through quarantine regulations.

Extensive efforts were therefore aimed at economic Avoid damage by, for example, planting plants on pathogen-free areas was carried out after the soil had previously been examined to this effect. this applies in particular with regard to the Agrobacterium tumefaciens Smith et Townsend Conn, who is a common inhabitant of cultivated soils all over the world. It attacks numerous cultivated plants and produced on the roots, on the root neck and above ground Organs Tissue growths known as "root heads", also as "Mauke", "Mauche", "Grind", English to be called "crown galis". 1), 2) The host plant circle of Agrobacterium According to ELLIOT 3) tumefaciens comprises 61 plant families with 140 species. In absence of hosts, the bacterium is able to survive saprophytically in the soil for several years. The infection takes place via wounds, which are caused by natural processes or by cultivation Measures arise. In the course of the summer half-year appear in dependency the weather conditions after incubation times of different lengths, the tumors.

1) Bergey's Manual of Systematic Bacteriology Vol. 1, Ganus Agrobacterium, 24ob254, Williams et al. Wilkins 1984.

2) The Prokaryotes, Von.1, Agrobacterium tumefaciens, 842-843, 846-851, Springer-Verlag 1981.

3) ELLIOT, C .: Manual of Bacterial Plant Pathogens.

2nd Ed., Chronica Botanica 186 pp., 1951.

As described by MoCF1E4) et alii, the tissue growths but also only start in the following growing season.

For trade, the plants must be national and international Requirement to be free from root goiter. Infestation means especially for nurseries significant economic losses; if necessary, buyers of infected goods are also damaged.

The simplest and at the same time safest method of getting an infection It is still important to avoid growing the plants on soils that are free are from the pathogen. Evidence has so far been limited to two methods.

Catch plant method according to STAPP 5). On areas intended for cultivation so-called catching plants were used and after the end of the growing season on the typical disease symptom (root crop) rated. STAPP recommended for this purpose Wild pears, which are particularly vulnerable.

Method by means of cultivation on selective nutrient substrates and subsequent Pathogenicity testing. Selective nutrient substrates allow due to their composition the growth of certain organisms, while the growth of other organisms is inhibited remain. There are several such nutrient media for isolating agrobacteria from soils (SCHROTH et alii6) CLARK 7 KADO and HESKETT 8) Soil samples taken, shaken in sterile water in the laboratory and determined by the suspension Quantities applied to the nutrient substrate.

4) MOORE, L.W. ANDERSON, A. and KADO, C.I .: Agrobacterium.

Lab. Guide Identification Plant Pathogenic Bacteria.

At the. Phytopathol. Soc., St. Paul, Min., 17-25, 1980.

5) STAPP, C .: Plant Pathogenic Bacteria. Agrobacterium tumefaciens 69-101, Parey 1958.

6) SCHROTH, M.N., THOMPSON, G.P. and HILDEBRAND, D.C .: Isolation of Agrobacterium tumefaciens-A. radiobacter group from soil. Phytopathology 55, 645-647, 1965.

7) CLARK, A.G .: A selective medium for the isolation of Agrobacterium species. J.Appl. Bacteriology 32, 348-351, 1969.

8) KADO, G.I. and HESKETT, M.G .: Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopathology 60, 969-976, 1970.

After incubation, shape, color and texture develop characterized bacterial colonies. However, the agrobacteria are different not to be distinguished, so that a pathogenicity test is used to detect A. tumefaciens must be connected. For this purpose, bacterial material is inoculated from the colonies on suitable test plants, which are then kept in the greenhouse. Does it around the pathogen, the growths form 2-4 weeks after inoculation.

In the event of positive results, the cultivation on the areas would be spaced apart or the soil would have to be chemically or physically decontaminated.

The procedures described are complex and involve factors of uncertainty burdened. It is true that they were developed with the aim of being useful in cultivation practice but the application is essentially more scientific to investigations Kind remained limited.

The disadvantages of the catch plant method are as follows. Already the Procurement of wild pears encounters difficulties because they have not been traded will; cultivation from seeds takes one growing season. use then find them 1-2 years old. Until the evaluation can finally be carried out, another up to 5 months pass and there is a dependency on the season and Weather.

In the detection by means of selective nutrient substrates, the narrow has an effect Relationship of the pathogenic and non-pathogenic agrobacteria is very disadvantageous. Their colonies on the artificial nutrient substrate are indistinguishable, so that a pathogenicity test is essential. According to information from SCHROTH et alii) is the Ratio between the bacterial counts of Agrobacterium radiobater (apathogenic) and A. tumefaciens (pathogenic) in the soil about 400: 1, based on our own experience 100-500: 1. A corresponding number of colonies are to be subjected to the pathogenicity test. If there are numerous soil samples to be examined, e.g. from large cultivation areas, the workload would hardly be manageable. It should also be taken into account that in the cultivation of the bacteria on the nutrient-rich artificial substrate a loss of virulence cannot be ruled out, which means that the test plants show no reaction.

After all, the method demands a certain amount from the practitioner Exercise in bacteriological work techniques.

The invention is based on the object of a method of the above mentioned genus in such a way that it is less expensive and safer and that clear test results are available in a much shorter period of time.

According to the invention, this object is achieved in that the aqueous Phase (shaking solution) of a shaken soil sample to remove the soil particles through a paper filter, then the shaking solution, which has been cleaned of soil particles with the help of a vacuum filter device through a membrane filter of the appropriate pore size sucks and then cuts the membrane filter into strips that you then at least a strip is inoculated into a test plant and the test plant after one of the Development characteristics of the microorganism corresponding to the incubation period the reaction of the test plant tissue checked.

In such a method, a disadvantageous intermediate cultivation avoided on artificial nutrient substrates by separating the bacteria through filtration will.

The procedure is described below in the process steps' Soil sampling and subsequent 'separation' of the bacteria by filtration.

From areas or from growing media that are used for the cultivation of plants, which belong to the host plant group of the pathogen, are provided according to Scheme Soil samples taken, each about 500 g or more. The withdrawal up to about the depth of the plow can be done with a sampler or a hand shovel. The samples are brought to the laboratory, mixed, if too wet, in the air beforehand dried. Then 10 g are weighed and autoclaved in a glass flask in 200 ml Saline was shaken on a shaker at 100 rpm for 1 hour. The number of revolutions can be increased so that the soil particles are moved well. A large proportion of the microorganisms present in the soil go during the shaking process into the aqueous phase (NaCl solution). After having stopped shaking the soil has settled on the bottom of the glass bulb, the supernatant will through put a pleated filter (20-24 cm) to remove soil particles. Then it takes place filtering out the bacteria.

Add 100 ml of the shaking solution to a suitable vacuum filtration device sucked through a membrane bacterial filter. In the case of Agrobacterium tumefaciens a filter with a pore size of 0.2 p is used because the cells of this bacterium have the dimensions 1.0 - 3.0 x 0.4 - 0.8 p and therefore remain in the filter.

The further process step of a pathogenicity test is as follows carried out: The filter is sterile in 4 strips or sectors cut up to be inoculated into the test plant. Any drying out is closed avoid; all operations must be carried out under sterile conditions.

In the case of bacteria of the genera Agrobacterium and Erwinia will the pathogenicity test was carried out on tomato plants that are grown in the greenhouse are. The plants are used in a size of 40-60 cm. The stalk will using a scalpel or comparable knife in 4 places at a distance of Slit open about 5-6 cm lengthways and use tweezers to fold them 4 strips / sectors of the bacterial filter placed in the wounds, these infection sites then covered with parafilm by wrapping. After that are the tomato plants to decapitate and fertilize. The (full) fertilization is repeated at weekly intervals. The greenhouse temperature should be 22-23 ° C (+ CO C) and not overnight sink too low.

If the tests are carried out during a low-light period Additional lighting displayed. 3 - 4 weeks after the inoculation are positive Reaction on the tomato stalks in the area of the inoculation sites, typical tissue growth recognizable. The cut surfaces become apart from the developing growths pressed. A negative finding is present if the wounds are after weak wound necrosis heal without growths appearing. A magnifying glass should be used for assessment will. The tissue overgrowth is evidence of Agrobacterium tumefaciens infection; thus the pathogen is present in the soil examined. There is a strong contamination before, there are correspondingly many pathogen cells in the bacterial filter and all of them four infection sites on the stem of the test plant respond positively; at lower Contamination, tumor formation can be limited to one site of infection. In model tests could be determined that the detection limit with the method shown with approx. 101-102 pathogen cells / g soil can be assumed. It is recommended to have a positive and to add a negative control. One of the purposes is the bacterial filter with a suspension of the highly virulent strain II b of Agrobacterium tumefaciens, the is easy to cultivate on nutrient agar, soaked, on the other hand it becomes a bacteria-free Filters placed in the stems of the tomato plants created wounds.

In the case of the Erwinia carotovora bacterium, the stem tissue also softens Blackening.

The detection method according to the invention is less complex and essential safer than the previous ones.

There is practically no dependence on the weather and the season. The tomato functioning as the test plant can be easily extracted from commercially available seeds are used, is fast-growing and without difficulty in greenhouse conditions to keep optimal. In comparison with the method of detection of pathogens via selective There is no difference in nutrient substrates when it comes to taking soil samples, However, the laboratory effort is then significantly lower. With the immediate Use of the bacterial filter for the pathogenicity test eliminates the "intermediate cultivation" the bacteria on the artificial nutrient substrate and the need to hedge the result of several hundred bacterial colonies per soil sample for pathogenicity to have to check. In addition, the possibility of a loss of virulence (infectivity) turned off the cultivation of bacterial pathogens on nutrient-rich Substrates in general. The new procedure demands from the implementers not necessarily experience in sophisticated bacteriological work techniques. A A particular advantage would result if based on a clearly negative result chemical disinfection of the intended floor area could be dispensed with.

This then means considerable savings in terms of medium and operating costs.

Description of two specific examples: 1. A horticultural company (e.g. tree nursery) grafts several million roses every year. Corresponding quantities of wild roses used as supports must be used will; one reckons around 40,000 wild roses, yes ha. The operation can take the risk of a stronger infestation and thus damage, or for safety reasons beforehand an expensive chemical soil disinfection (e.g. with methyl bromide, Shell-DD, DiTrapex, Basamid). There is now the possibility of using the described Method the soil for the presence of the root goiter for a short time at relative check with little effort. The result is an important aid in deciding whether the floor area can be used for cultivation or not, or whether a soil disinfection must be undertaken or whether it can be dispensed with 2. A horticultural company (e.g. ornamental plants sector) produces outside of Germany or Europe Chrysanthemum cuttings (young plants) in very large quantities, in batches to sell to other nurseries who cultivate the plants until they bloom and market. If the cuttings (young plants) were infected by the root goiter pathogen, If the cultivation continues, the tumors develop and the plants do not develop to sell (to be marketed). The buyer of the cuttings (young plants) is of the Producers claim damages. The latter would now be able to use the method described to check the cuttings (young plants) for non-infestation. He only dispatches healthy goods and does not have to fear any claims for damages.

The method can be used for the detection of further significant in the soil and microorganisms common to plants.

The following examples are given for this: bacteria Genus Agrobacterium Agrobacterium rhizogenes, causative agent of the "hairy root" disease on rose plants, cell dimensions 1.5 - 3.0 x 0.6 - 1 µ; Membrane filter with 0.2 pore size; Test plant Tomato plants genus Erwinia Erwinia carotovora, causative agent of wet rot on various plants and fruits, cell dimensions 1.5 - 2.5 x 0.6 - 0.8 p; Membrane filter with 0.2 p pore size; Test plants Tomato plants Erwinia amylovora, causative agent of the Fire blight disease of the rose family, cell dimensions 0.9 - 1.8 x 0.6 - 0.9 p; Membrane filter with 0.2 µ pore size; Test plant pear fruits genus Corynebacterium Corynebacterium sepedonicum, causative agent of bacterial ring rot in potatoes, cell dimensions 0.6 - 1.4 x 0.3-0.5 µ; Membrane filter with 0.2 pore size; Test plant potato tubers mushrooms Parasitic slime molds and chytridiomycetes e.g. Plasmodiophora brassicae, pathogens the hernia of cabbage, cell dimensions> 1 µ; Membrane filter with 1, u pore size; Test plant Kohlrabi.

Claims (4)

Method for the detection of phytopathogenic microorganisms in the soil and in infested plants. Claims 1. Biological process for the detection of phytopathogenic Microorganisms in the soil and in infested (suspected infestation) plants, in particular of bacteria of the genera Agrobacterium and Envinia as well as parasitic slime molds and Chytridiomycetes, characterized in that the aqueous phase (shaking solution) a shaken soil sample to remove the soil particles through a paper filter there, then the shaking solution cleaned of soil particles with the help of a vacuum filter device Sucks through a membrane filter of the appropriate pore size and then the membrane filter cut into strips that at least one strip is then cut into a test plant inoculated and the test plant according to one of the development characteristics of the microorganism appropriate incubation time checked for the reaction of the test plant tissue. 2. The method according to claim 1, characterized in that one slit open the tissue of the test plant for inoculation, the moist test strip lays in the slit wound and closes it with a bandage. 3. The method according to claim 1 or 2, characterized in that that the test strip is inoculated immediately after the end of the vacuum filtration. 4. The method according to any one of the preceding claims, characterized characterized in that the inoculated test plant or the inoculated test plant part during the incubation period under for the development of the pathogenic microorganism maintains optimal temperature conditions.