Method for the isolation of symbiotic microorganisms from host plants using an osmium tetraoxide fixative

Symbiotic microorganisms such as nitrogen fixing bacteria and phosphorus-providing fungi are isolated from nodules and mycorrhiza on the roots of host plants by sterilizing the surface and subsurface areas of root tissue with an aqueous solution of a fixative containing osmium tetraoxide. The osmium tetraoxide is highly soluble in water, and volatilizes at room temperature to form a vapor which penetrates subsurface air pockets in root tissues to kill contaminating microorganisms. The osmium tetraoxide also provides a visual indicator of its progress through plant tissue by a progressive darkening of such tissue from the surface of root samples toward their centers. In this manner, the sterilization can be halted before the centrally located causitive bacteria is destroyed.

BACKGROUND OF THE INVENTION 
This invention relates to a process for the isolation of endosymbiont 
nitrogen-fixing bacteria and phosphorous-providing fungi in the roots of 
plants, and more particularly to an improved surface sterilization process 
for such roots to isolate the bacteria and fungi. 
Nitrogen and phosphorus are essential mineral nutrients for plants. A 
shortage or lack of either nutrient in soils will adversely affect plant 
growth. Heretofore, nitrogen and phosphorus have been supplied to crops 
and other bromass producing plants by the addition of fertilizers to the 
soil. However, increasing costs of producing such fertilizers have led to 
searches for alternative methods of supplying these mineral nutrients to 
growing plants. 
It is known that nitrogen-fixing root nodules occur on both leguminous and 
non-leguminous plants. Members of the legume family whose roots are 
infected by the soil bacterium Rhizobium are the primary source of 
nitrogen fixation in agricultural systems. Some non-leguminous angiosperms 
form root nodules when invaded by soil actimomycetes bacteria which also 
enable fixation of atmosphere nitrogen by these plants. Additionally, it 
is known that certan fungi produce ectomycorrhizal and endomycorrhizal 
growths on plant root systems which can provide phosphorus to several 
plant species including some which are agriculturally important. 
Presently, the successful growth of nitrogen-fixing bacteria or 
phosphorus-providing fungi on a particular host plant depends on the 
presence of an endogenous population of such bacteria or fungi in the soil 
in which the plant is growing. When planted in soils having a low 
concentration of mineral nutrients, the absence of early and effective 
growth of root nodules and/or mycorrhiza delays the establishment and 
growth of seedlings and may even result in their complete failure. 
Previous methods of inoculating seedlings with suspensions of ground-up 
nodules or applying soil suspensions taken from where host plants were 
growing gave unpredictable and variable results because such suspensions 
contained a wide range of soil microorganisms as well as complex products 
derived from broken plant tissues. 
Clearly, the need exists for techniques which will enable the isolation and 
cultivation of the causative bacteria and fungi which can then be used to 
inoculate seeds and/or seedlings. While isolation and identification of 
Rhizobium bacteria from legume root nodules have been known for sometime, 
only recently have successful isolation techniques been developed for 
certain non-leguminous plants. See, Callaham et al., Science, volume 199, 
pp. 899-902 (1978). Such techniques include surface sterilization of root 
nodules using sodium hypochlorite or mercury chloride solutions followed 
by microdissection and enzymetic digestion or suspension and dilution of 
the host tissues. Other reported techniques for isolating such bacteria 
from root nodules include Sephadex and sucrose density fractionation. 
However, these techniques have not proved to be entirely satisfactory. Not 
only are they time consuming and technically complex, they exhibit a very 
high failure rate in successfully isolating the causative bacteria. 
Presently utilized surface sterilization techniques, designed to kill the 
wide variety of other soil microorganisms found on root nodules while 
leaving unaffected the causative bacteria and fungi, have not been 
successful. This is due to several factors. One factor is that air pockets 
and small pathways in the interior of such root nodules harbor a variety 
of microorganisms which the surface sterilant does not reach. When the 
root nodules are crushed or dissected, these microorganisms are 
transferred along with the desired endophytes to a cultivation medium, 
causing contamination of the medium. Another factor is that the surface 
sterilants heretofore used in many cases act so quickly on the root 
nodules and mycorrhizal growths that all of the desired microorganisms are 
killed along with the rest of the microorganisms. 
Accordingly, the need exists in the art for an improved isolation technique 
for nitrogen-fixing bacteria and phosphorus-providing fungi which is both 
simple to perform and results in a high success rate in providing an 
isolated culture. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, symbiotic microorganisms such as 
bacteria and fungi are isolated from nodules and mycorrhiza on the roots 
of host plants by first sterilizing the surface of the roots with an 
aqueous solution of a fixative containing osmium tetraoxide. The osmium 
tetraoxide is effective in killing microorganisms, is highly soluble in 
water, and volatilizes readily at room temperature to form a vapor. This 
volatility aids in allowing the osmium tetraoxide to penetrate subsurface 
air pockets in root tissue and kill contaminating microorganisms. 
Moreover, because of the relatively large size of the osmium atom, and thus 
the osmium tetraoxide molecule, the molecule penetrates plant tissue very 
slowly as compared to prior art fixatives used as surface sterilants. 
Because of this slow penetration of root tissue, the sterilization of 
roots of sample plants can be controlled. That is, the osmium tetraoxide 
can be used to kill all surface and subsurface contaminating 
microorganisms and then be rinsed from the root sample before it has 
penetrated to the core of the sample. This permits the symbiotic bacteria 
and fungi of interest to survive and be isolated. 
An additional advantage in the use of osmium tetraoxide in the practice of 
the invention is that as it contacts plant tissue, some of the tetraoxide 
compound reacts to form osmium hydroxide, a visually black precipitate. 
Thus, the optimum treatment time for root samples can be easily determined 
by a visual observation of the progressive darkening of the root tissue 
from its periphery toward its center. Once the osmium tetraoxide has 
penetrated sufficiently below the surface of the sample to kill all 
contaminating microorganisms, the sterilization can be stopped, based on 
visual observations, and the endophytic symbiotic bacteria and fungi can 
be recovered alive. 
Once recovered, these symbiotic bacteria and fungi are then transferred to 
an appropriate growth medium where sufficient quantities may be grown for 
use as inoculants for other host plants. Such plants can be inoculated 
using known techniques such as applying an aqueous suspension of the 
symbiotic bacteria or fungi to host plant seeds or to the soil surrounding 
host plant seedlings. In this manner, the host plants are provided with 
sources of nitrogen and/or phosphorus from their symbiotic relationship 
with bacteria and/or fungi even in mineral-poor soils without the need for 
the application of expensive fertilizers. 
Accordingly, it is an object of the present invention to provide an 
improved technique for isolating nitrogen-fixing bacteria and 
phosphorus-providing fungi from host plants which is both simple and has a 
high success rate in providing an isolated culture. This and other objects 
and advantages of the invention will be apparent from the following 
description and appended claims. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In accordance with the practice of the present invention, fresh root tissue 
samples containing nodules or mycorrhiza can be collected directly from 
field-grown and/or greenhouse-grown plants. The isolation technique of the 
present invention is useful in separating several types of both 
nitrogen-fixing bacteria and phosphorus-providing fungi. 
For example, soil actinomycetes, such as various Frankia strains, which 
nodulate the root systems of nonleguminous angiosperms are known 
nitrogen-fixing endophytes. Rhizobium bacteria are also known to form root 
nodules and fix nitrogen in legumes. Several strains of 
phosphorous-providing fungus induced ecto-and endomycorrhizae also are 
known to exist in symbiosis with agriculturally important plants. All of 
these types of symbiotic microorganisms can be isolated using the 
technique of the present invention. 
Root tissue samples containing the desired bacteria or fungi are collected 
and small pieces (i.e., 2-5 mm in length) are then immersed in a fixative 
solution containing osmium tetraoxide. Typically, the amount of osmium 
tetraoxide in solution can vary from 1-5%, by weight with 3% by weight 
being preferred. Lesser and greater strength solutions can be used, 
although the time need to fix effectively the contaminating surface and 
subsurface organisms in the tissue samples will need to be varied 
depending upon the concentration of the solution and the size and type of 
samples. The greater the concentration of osmium tetraoxide, the faster 
the sterilization proceeds. Generally, a 3% osmium tetraoxide in 0.05 M 
phosphate buffered saline solution having a pH of about 7.2 with 0.6% by 
weight calcium chloride has been found to be effective when contacted with 
tissue samples for from 30 seconds to 6 minutes. Tissues are fixed at room 
temperature. 
Osmium tetraoxide is a highly potent fixative which is readily soluble in 
water and tends to volatilize at room temperature. Accordingly, it is the 
preferred fixative for the present process, although, other fixative 
materials could also be used if they had the same characteristics as 
osmium tetrooxide, including the ability to volatilize at room 
temperature. The vaporization of osmium tetraoxide aids in its penetration 
into subsurface air pockets to kill contaminating microorganisms. However, 
because of its toxic vapors, care should be taken when using it; good 
ventilation and a fume hood is recommended for handling solutions. 
Another advantage in the use of osmium tetraoxide is that its optimum 
fixation time for tissue samples can be readily determined by a visual 
observation of the progressive darkening of the tissue samples from the 
periphery toward the center caused by the formation of osmium hydroxide. 
Since the object of the isolation technique is to preserve at least some 
of the desired bacteria or fungal strains alive, sample root tissue should 
be fixed only to the extent of killing surface and subsurface organisms. 
This technique works well with the endophytic bacteria and fungal strains 
and also works to isolate some ectomycorrhizal fungi because at least a 
portion of the desired species can be found in the interior of a tissue 
sample. 
The sterilization procedure is terminated by removing the tissue samples 
from the osmium tetraoxide solution and rinsing them in sterile water. The 
rinsing procedure should be repeated one or more times to insure that all 
traces of fixative are washed away. The water-rinsed tissue samples are 
then immersed aseptically in a sterile solution containing an appropriate 
buffer solution such as a 0.05 M phosphate buffered saline (0.6% NaCl, 
pH=7.2) which also contains approximately 0.1% W/V of soluble 
polyvinylpyrrolidone. The use of polyvinylpyrrolidone in the buffer 
immobilizes phenols contained in some plant tissues. 
After immersion for a few minutes in the buffer solution, the tissue 
samples are divided into several pieces (i.e., 2-6 pieces per sample) 
using an appropriate sterile tool such as a needle, razor blade, or 
scalpel. Each piece is then transferred aseptically to an appropriate 
sterile liquid or gelled growth medium. One growth medium which has been 
found to suitable for cultivation of numerous endophytes is a growth 
medium identified as Qmod by Lalonde and Calvert, "Production of Frankia 
Hyphae and Spores as an Infective Inoculant for Alnus Species," Symbiotic 
Nitrogen Fixation in the Management of Temperate Forests, Workshop held 
Apr. 2-5, 1979, Oregon State University, Corvallis, Oreg. This Qmod growth 
medium has the following constituents: 
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per liter 
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K.sub.2 HPO.sub.4 300 mg 
NaH.sub.2 PO.sub.4 200 mg 
MgSO.sub.4.7H.sub.2 O 200 mg 
KCl 200 mg 
Yeast Extract (BBL) 500 mg 
Bacto-Peptone (DIFCO) 5 g 
Glucose 10 g 
Ferric Citrate 
(Citric Acid and Ferric Citrate, 1% sol.) 
1 ml 
Minor Salts* 1 ml 
H.sub.2 O, Deionized to: 1 l 
adjust pH to 6.8-7.0 with NaOH or HCl 
then add, CaCO.sub.3 100 mg 
Lipid Supplement** 0.5-50 mg 
Agar, if used 15 g 
Mix thoroughly and pour 15 ml per tube, autoclave for 20 
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min. 
*Minor Salts (g/liter): H.sub.3 BO.sub.3, 1.5; MnSO.sub.4.7H.sub.2 O, 0.8 
ZnSO.sub.4.7H.sub.2 O; 0.6; CuSO.sub.4.7H.sub.2 O, 0.1; (NH.sub.4).sub.6 
Mo.sub.7 O.sub.24.4H.sub.2 O, 0.2; CoSO.sub.4.7H.sub.2 O, 0.01. 
**Lipid Supplement: dissolve 500 mg of Llecithin (commercial grade from 
soybeans, 22% phosphatidyl choline, P5638 from Sigma Chemical Co., St. 
Louis, MO) in 50 ml of absolute ethanol, and add 50 ml of distilled water 
 
The inoculated growth medium is then incubated at from 
15.degree.-27.degree. C. for from several days to a few weeks until a 
sufficient growth of the isolated bacteria or fungus is observed. This 
bacteria or fungus can then be inoculated onto seeds or seedlings to 
infect them with the desired symbiotic nitrogen-fixing or 
phosphorus-providing culture. The success of the isolation technique of 
the present invention has been found to average more than 50%. That is, 
more than 50% of the pieces isolated by the technique, when cultured, 
produce pure cultures of the desired microbial isolate.

The following nonlimiting example is provided to illustrate the practice of 
the present invention. 
EXAMPLE 
Root nodule samples containing a Frankia strain of nitrogen-fixing soil 
actinomycetes were collected from an Alnus serrulate host plant growing in 
soil in a green house. The nodule samples were promptly treated in 
accordance with the practice of the present invention. The samples were 
immersed in a 3% solution of osmium tetraoxide for periods varying from 2 
to 5 minutes. The samples were then rinsed in sterile water followed by 
immersion in a phosphate buffered saline solution which contained 
polyvinylpyrrolidone. 
The samples were then cut into 50 pieces and incubated individually in 50 
separate test tubes (20.times.150 mm) into which 17 ml of Qmod growth 
medium had been added. After one week, 28 of the test tubes were observed 
to contain pure cultures of a Frankia strain isolate, indicating a 56% 
success rate. 
While the processes and compositions herein described constitute preferred 
embodiments of the invention, it is to be understood that the invention is 
not limited to these precise processes and compositions, and that changes 
may be made therein without departing from the scope of the invention.