Process for inclusion of mycorrhizae and actinorhizae in a matrix

This invention relates to a process of inclusion of microorganisms of the group consisting of mycorrhizae and actinorhizae in a polymer gel matrix to prepare a solid, stable, storable preparation suitable for use in particular for agronomic purposes. The polymer gel matrix is based on at least one polymer from the polysaccharide group, with at least partial cross-linking of the polymer.

BACKGROUND OF THE INVENTION 
The invention concerns a method of inclusion of microorganisms of the group 
consisting of mycorrhizae and actinorhizae in a matrix comprising a 
polymer gel, to prepare a product suitable for use in particular for 
agronomic purposes. 
It was proposed many years ago to fix microorganisms on a carrier in U.S. 
Pat. No. 1,909,622, in which nitrogenfixing bacteria of the genus 
Rhizobium are used. 
Generally, however, the microorganism culture is adsorbed onto a carrier, 
as in Belgian patent 521,850, which advocates the use of diatomaceous 
earth and colloidal silica for Rhizobium. Bentonite has also been used (in 
British Patent No. 1,777,077), as have plaster granules (in French Patent 
No. 1,490,046) and even lignite. 
This method of inclusion by adsorption has disadvantages, particularly in 
connection with the survival of the microorganism and its protection 
during transport, storage and handling. Since this procedure gives only 
very limited results, attempts have been made to improve fixing 
techniques. 
Thus, French Patent No. 1,180,000 calls for the use of a must juice, to 
which substances with an adsorbing action are added, such as cellulose, 
bone meal, kaolin or silica gel, in the manufacture of preparations rich 
in bacteria of the Azotobacter group. This type of preparation is also not 
satisfactory. 
Attempts have therefore been made to improve both the survival rate of the 
microorganisms, e.g., by inclusion, and the manner of bringing them into 
the plant medium. Thus, in French Patent application No. 77.10254 of Apr. 
5, 1977 (corresponding to U.S. Pat. No. 4,155,737) a method is proposed 
which makes use of an inoculum comprising a polymer gel with microorganism 
included therein. The inoculum is introduced into the rhizosphere of the 
plants. According to this patent, the polymer gel may be a polyacrylamide 
gel or a silica gel. 
None of the above methods has been entirely satisfactory in meeting all of 
the requirements for an adequate carrier. Such a carrier must maintain the 
microorganism under conditions sufficient to preserve, protect and keep it 
in a suitable form for handling, while at the same time enabling it to 
swarm into the medium and if possible allowing for the grafting of 
additives onto the carrier. Furthermore, the carrier must insure the 
viability of the microorganism, even after periods of several weeks and 
under conditions of variable hygrometry. This means that the carrier must 
be able either to contain a sufficient reserve of water, which can be made 
available as necessary, or to obtain the necessary water from the 
environment. Finally, the carrier must not be detrimental to the 
environment, i.e., it must be either biodegradable or non-polluting. 
This rapid enumeration of requirements, which makes no claim to be 
exhaustive, suggests why until now a suitable method has not been 
developed as expected. 
In French Patent application 79.08597 of Apr. 5, 1979, there is disclosed a 
particularly attractive method, comprising the inclusion of the 
microorganism in a polymer of the polysaccharide group and an at least 
partial cross-linking of the polymer, e.g., by heat treatment, through use 
of a metal salt or through synergism with another polymer. This method 
gives surprising results, and a remarkable synergism is shown when a 
compound which is absorbent and adsorbent, such as a silica, is also used. 
The above-noted French patent application is directed in particular to 
preparations of Rhizobium japonicum, a non-sporulated, nitrogen fixing 
bacterium which is very sensitive to drying, temperature and 
physico-chemical factors. Some microorganisms, however, present additional 
difficulties because of their fibrous nature, such as ectomycorrhizian 
fungi (e.g., Pisolithus, Hebeloma, Tuber, Boletus, etc.) which, moreover, 
have no form of resistance to, e.g., pathogenic germs, when cultivated in 
pure culture. 
Mycorrhizian associations are the result of associating a fungus with a 
root, leading to a true symbiosis. The following groups are distinguished 
by the nature the association: 
(a) ectotrophic mycorrhizae, found chiefly in woodland trees (Pinaceae, 
Fagaceae), most being upper fungi (Ascomycetes and Basidiomycetes); and 
(b) endotrophic mycorrhizae, usually lower fungi (Phycomycetes), much more 
common than the above and found in trees, shrubs and herbaceous plants in 
the case of mycorrhizae with arbuscles and vesicles, but limited to 
Ericaceae in the case of mycorrhizae with clusters. 
The beneficial action of these mycorrhizae on the growth of plants can be 
attributed to plant hygiene protection against pathogens in the ground, 
production of growth-promoting substances or vitamins improvement in the 
mineral nutrition of the plant (particularly with respect to phosphorous) 
through an increase in the possibilities for ground exploration, and 
improvement in water absorption where there is a shortage of water. 
In the case of nitrogen-fixing non-leguminous plants, symbiotic 
associations of the same type as those existing between Rhizobium and 
leguminous plants are characterized by the formation of nodules on the 
root system. These are found both in trees and shrubs and in herbaceous 
plants. The function of the nodules is known only in certain ligneous 
plants which generally colonize poor or degraded soils (sands, moraines), 
where real fixation of atmospheric nitrogen has been shown to take place. 
137 species of Angiospermae belonging to 12 different genera, classed in 
seven families (Betulaceae, Casuarinaceae, Coriariaceae, Eleagnaceae, 
Myricaceae, Rhamnaceae, Rosaceae) have been recognized (Bond 1974). The 
endophyte responsible for forming the nitrogen-fixing nodules is an 
Actinomycetum (Frankia) which had only been isolated in pure culture in 
1978 by Lalonde et al. (Laval University, Quebec). 
There is no doubt of the importance of using these nitrogen-fixing 
varieties of microorganisms in sylviculture, particularly in marginal 
soils which are poor in nitrogen and with a modified structure or none at 
all. The following examples can be given as an indication of typical uses: 
(1) reforestation of peat bog in France (Aulne, Myrica), glacial moraines 
in the Alps (Aulne), soil thrown up in mining or quarrying oil shale in 
the U.S.A.; 
(2) fixing maritime and continental dunes in Senegal (Casuarina); 
(3) developing James Bay in Canada; 
(4) Alnus rubra - Douglas associations in the N.W. American forest systems; 
(5) use of Alnus glutinosa, A. cordata, A. incana, A. crispa as nurse trees 
to encourage development of nonfixing species; 
(6) use of Ceanothus, Myrica, Hipophaca, Eleagnus in association with 
non-fixing species or in production of green fertilizer or biomass. 
Inoculation has been traditionally carried out at the nursery stage, using 
crushed nodules. This has the serious disadvantages that pathogenic germs 
may be introduced into the inoculum and that the nodules cannot be 
preserved, even at low temperature, because of the rapid oxidation of 
tannins and phenolic substances, which are toxic to the microorganisms. It 
has not thus far been possible to demonstrate in a formal manner that the 
productivity of these species can be increased by inoculating with an 
endophyte developed in pure culture. 
With ectomycorrhizae, inoculation has traditionally been carried out using 
soil from another nursery; currently, it is more common to use pure 
cultures of mycorrhizian fungi in vermiculite. Inoculation carried out in 
this way has three major disadvantages: 
(1) the difficulty in developing the fungus (e.g., Pisolithus or Hebeloma): 
At least six weeks time is required (Grahan-Linderman 1980 Can. J. Microb. 
26, II) to obtain a thin, heterogeneous culture in a liquid medium; at 
least eight to ten weeks is required on vermiculite. In the case of a 
liquid culture, it is necessary to recover the mycelium and crush it 
before use, with the dangers of excessive shearing leading to no regrowth. 
This is particularly true for Tuber, since the microorganism has only 
hyphae and no form of resistance. 
(2) the large quantity of inoculum required: 2 l/m.sup.2 in the case of 
microorganisms developed on vermiculite, with the corresponding difficulty 
in storage; and 
(3) the need to inoculate with inocula which have been freshly prepared or 
stored at 4.degree. C. It is these problems which have led to the 
development of the instant invention. 
One object of the invention is to develop an inoculum which can be stored 
at ambient temperature and is easy to use. 
Another object is to improve the properties of cultures of mycorrhizae and 
actinorhizae, particularly with respect to the level of homogeneity. 
Still another object of the invention is to achieve a reduction of 
cultivation time, in particular for actomycorrhigae 
GENERAL DESCRIPTION OF THE INVENTION 
The present invention is concerned with a process of inclusion of a 
microorganism from the group consisting of mycorrhizae and actinorhizae in 
a matrix comprising a polymer gel based on at least one polymer from the 
polysaccharide group, with at least partial cross-linking treatment of the 
polymer. 
In accordance with the invention the "at least partial cross-linking 
treatment" is understood as being a treatment which can modify the 
structure of the polysaccharide, such as heat treatment, treatment with a 
metal salt or a synergistic treatment using a different polymer and 
preferably a different polysaccharide. 
The polymer is advantageously based on a heteropolysaccharide of high 
molecular weight, obtained by fermenting a carbohydrate with a 
microorganism of the genus Xanthomonas or Arthobacter or with fungi of the 
genus Sclerotium. 
Polymers obtained from natural or biosynthetic gums from various sources 
may also be used, for example: algae (alginates, carrageenans, agar), 
exudates of plants (gums such as karaya, tragacanth, arabic) or seeds 
(guar, carob). 
Locust bean gum is an extract from the fruit of the carob tree (Ceratonia 
siliqua L.), a tree of the family Caesalpiniaceae (geographical area: 
Mediterranean basin). Locust bean gum is contained in the endosperm of the 
seed. The endosperm is separated from the embryo by mechanical abrasion or 
a chemical process. Locust bean gum is a polysaccharide (galactomannan) 
consisting of .beta.-D-mannopyranosyl units (bonds 1-4), one out of four 
or five being substituted at C.sub.6 by .alpha.-D-galactopyranosyl. 
The combination of xanthan gum with locust bean gum by a synergistic effect 
increases the viscosity of the gel. It is believed that the xanthan gum is 
made up of helical chains and that interaction with galactomannan leads to 
the formation of bonds between the chains, enabling a gel with a 
three-dimensional network to be obtained. 
The alginates are refined extracts of brown algae of the Phaeophyceae 
class. Alginic acid is a straight polymer of high molecular weight formed 
by a succession of molecules of .beta.-D-mannopyranosyluronic acid and 
.alpha.-L-gulopyranosyluronic acid. The addition of calcium ions links two 
carboxyl groups, forming a bridge between parallel chains, particularly in 
the regions consisting of guluronic acid. In this manner, a gel of higher 
viscosity is obtained instantly at 25.degree. C. Conversely, the addition 
of phosphate ions enables solidification to be retarded; this may be 
helpful for handling the preparation before it is completely gelled. 
As indicated in French Patent application 79.08597 (European Patent 
application 17 565), the concentration of microorganisms in the 
preparation may be increased by filtering or centrifuging the culture 
medium, resuspending the filtrate or bottom in a small volume and adding 
it to the polysaccharide solution. 
The microorganism or microorganisms may be added in various ways. 
Generally, a culture medium is first prepared, which is seeded with the 
microorganism; the culture medium, or the suspension of the microorganism 
obtained by filtering and centrifuging, is then added to the 
polysaccharide solution; and a gel is finally formed by cooling. 
In particular, in a first embodiment of the invention, a polysaccharide 
solution is first formed hot and brought to a temperature of approximately 
40.degree. to 45.degree. C. The culture medium containing the 
microorganism, or the suspension of the microorganism, is then added and 
the mixture is cooled to form the gel. 
In another embodiment, the culture medium or microbial suspension is added 
separately to each polysaccharide solution under the same temperature 
conditions. The substances are then mixed and cooled to form the gel. 
As noted above, it is also possible to use a metal salt such as one of iron 
or aluminum, which may or may not be complexed with a polyol. 
Alternatively, the polysaccharide may be dissolved in the culture medium, 
particularly at ambient temperature, and the cross-linking carried out in 
situ. 
As previously mentioned, the microorganism of the invention may be an 
actinorhiza such as the endophytic actinomycetum of a non-leguminous 
plant, or a mycorrhiza, ectomycorrhiza or endomycorrhiza. 
The inocula may be in various forms: gel, powder, pellets or even fibers. 
The actinomycetum may be provided in the form of liquid culture or in the 
form of crushed nodules. 
It is advantageous to dry the gel. As already mentioned, the microorganism 
is known to be generally very sensitive to heat. Ordinary drying takes a 
long time and gives a dry film which crumbles easily and can be crushed 
without difficulty. It is therefore preferable to add an absorbent 
substance with a large capacity for absorbing water to the gel, so that 
the residual water content obtained in the gel plus absorbent mixture is 
from 70 to 250 g/100 g of absorbent and advantageously from 100 to 150 
g/100 g. 
The absorbent substance is a porous material such as natural or synthetic 
silica, silico-aluminates, cellulose, etc. The pH is in the region of 7, 
and the drying temperatures are low enough not to destroy the 
microorganism, e.g., on the order of 20.degree. to 30.degree. C. 
The material may be put into its final form in various ways. 
In a first embodiment, the gel is dried and then finely crushed, a 
substance such as silica is added to it and the two are homogenized. The 
resultant powder can then be put into tablet form. 
In another embodiment, the moist gel and the substance such as silica are 
placed in a mixer. After being worked, the mixture may be spread out and 
dried directly, until the loss of water is from 0 to 50% of its weight but 
preferably from 30 to 40%, giving a powder with a residual water content 
of from 100 to 150 g per 100 g of absorbent material. Alternatively, the 
mixture is placed in an extruder and the granules obtained are dried at 
ambient temperature, again until there is a loss of water of from 30 to 
40%. 
In cases where the microorganism is a mycorrhiza, this may advantageously 
be put into the form of a homogeneous mycelian culture. The inoculum may 
be in the form of moist pellets, threads or fibers. 
In a first embodiment, the volume of liquid culture may be converted to a 
substantially identical volume of solid, so that moist pellets can 
advantageously be obtained. In a second embodiment, the gel obtained when 
the culture or suspension of the microorganism has been included has an 
absorbent substance added to it, so as to give a powder which can easily 
be handled. The absorbent substance is advantageously a silica, its 
percentage by weight relative to the gel being from 10 to 120% and 
preferably 30 to 50%. 
In both embodiments, the process has the considerable advantage that the 
mycelium does not have to undergo any mechanical treatment such as 
shearing or crushing. In addition, the process provides inocula which can 
be stored at ambient temperature, under non-sterile, non-contaminating 
conditions. 
SPECIFIC DESCRIPTION OF THE INVENTION 
In order to disclose more clearly the nature of the present invention, the 
following examples illustrating the invention are given. It should be 
understood, however, that this is done solely by way of example and is 
intended neither to delineate the scope of the invention nor limit the 
ambit of the appended claims.

EXAMPLE 1: Endophytic actinomycetum of non-leguminous plant 
1. Stock - Isolation - Cultivation - Preservation 
Symbiosis studied: Alnus glutinosa - Frankia 
Stocks: Frankia ARbNN4b and AgNlag (Lalonde Collection, Faculty of Forestry 
and Geodesy, Laval University, Quebec). 
Isolation of Frankia from nodule (technique developed by Lalonde et al. 
Proc. Workshop 2.5/4-1979-Corvallis-Oregon). 
An end of a coralloid nodule is surface-sterilized (30% H.sub.2 O.sub.2 for 
5 minutes; then 5% NaClO for 30 minutes). It is rinsed with sterile water. 
The nodule is placed in a salt cellar containing a 1% solution, which is 
sterilized by filtering polyvinylpyrrolidone (PVP)+phosphate buffer (PBS 
(g/l)=NaCl, 0.8; Na.sub.2 HPO.sub.4 .multidot.7H.sub.2 O, 1.14; KH.sub.2 
PO.sub.4, 0.2). The lower part of the nodule is sectioned and the end 
transferred to a different salt cellar containing PBS (a few drops). The 
nodule is crushed and the inside is taken out and used to seed a test tube 
containing Q mod* medium (Quispel 1960, as modified by Lalonde 1979). 
*Q mod (g/l): K.sub.2 HPO.sub.4, 0.3; NaH.sub.2 PO.sub.4, 0.2; MgSO.sub.4 
.multidot.7H.sub.2 O, 0.2; KCl, 0.2; Yeast Extract (BBL), 0.5; 
Bactopeptone (DIFCO), 5; glucose, 10; ferric citrate (citric acid+ferric 
citrate solution 1%), 1 ml; oligo-elements, 1 ml; H.sub.2 O to give 1000 
ml (pH 6.8-7.0); CaCO.sub.3, 0.1; lecithin, 5 mg. Sterilization: 20 
minutes at 120.degree. C. The material is incubated for two weeks at 
27.degree. C. 
CULTIVATION 
After incubation, the mycelium is split up with a syringe and 6 tubes are 
reinoculated. The same procedure is carried out every two weeks, as a 
means of actively multiplying the Actinomycetum. Before each planting out 
process the culture is washed with PBS. The colony is taken from the 
bottom of the tube with a pipette and transferred to a Q Mod medium. 
PRESERVATION - GERMINATION OF SPORES 
Germination of spores in a culture which has been stored for several months 
is encouraged by immersing the colony in a solution consisting of 45 mg 
alanine+60 mg leucine+50 ml PBS for 30 minutes and by returning it to the 
Q mod medium after splitting it up. 
2. CULTIVATION OF PLANTS (ALNUS GLUTINOSA) 
A. Pregermination of seeds 
This operation includes four phases: 
(1) SELECTION OF SEEDS 
The seeds are placed in a beaker containing hexane (d=0.66). Those which 
drop to the bottom are taken out and dried on paper, and the floating 
seeds are discarded. 
(2) DORMANCY SPROUTING 
The seeds are placed in water and put at +4.degree. C. for 4 days. 
(3) STERILIZATION 
This is carried out in 5% NaClO for 5 minutes, followed by washing with 
sterilized distilled water. 
(4) PREGERMINATION 
The seeds are placed on a medium which has been treated with gelose, on a 
Petri dish, containing 1% of gelose and 1.5% of saccharose. The Petri 
dishes are arranged upside down in a chamber saturated with water, and are 
kept at 28.degree. C. in the dark for 6 days. 
B. Pricking out plantlets 
The non-contaminated plantlets are pricked out into "Pouches" or test tubes 
with a diameter of 22 mm. 
(1) Pricking out into "Pouches" 
Filter paper with a spout at the top is placed in a polyethylene bag 
(10.times.20 cm). 8 ml of Crone solution (as modified by Lalonde, J. Can. 
Botanic 50, 2597-2600 (1972)), without any nitrogen, diluted by 1/2 in the 
bag, is poured in so as to moisten the whole filter. Holes are made in the 
spout, and the plantlets are arranged with their rootlets facing the 
opening and so that the cotyledons extend beyond the spout. The bags are 
placed in a controlled room (lighting 15000 lux, photoperiod 14 hours, 
temperature from 19.degree. to 23.degree. C.). 
(2) Planting out onto vermiculite 
Tubes with a diameter of 22 mm are filled with 40 ml of vermiculite washed 
with water. 20 ml of Crone solution, diluted by 1/2, is added, after which 
the whole arrangement is sterilized for 20 minutes at 120.degree. C. The 
plantlets are pricked out and incubated as above. 
3. PREATION OF INOCULA 
3.1 INOCULA WITH INCLUDED FRANKIA CULTURE 
Frankia colonies obtained from 10 cultures in test tubes, which have 
developed for 15 days, are taken out, resuspended in 55 ml of PBS and 
split up. The inocula are prepared with this concentrated suspension, 
containing hyphae, spores and spore cases, as follows: 
INOCULUM BASED ON ALGINATE (AlG) 
The concentrations are given for the preparation of 45 g of gel. 0.45 g of 
alginate is dissolved in 36 ml of suspension; 9 ml of a 6 g/l solution of 
CaSO.sub.4 .multidot.2H.sub.2 O is stirred in. The gel obtained is then 
either dried in air until dehydrated, or 40% of its weight of silica is 
added and the two are mixed until a moist homogeneous powder is obtained. 
The powder is then dried at ambient temperature until the inoculum 
contains only 125 g of water per 100 g of silica. 
INOCULUM BASED ON XANTHAN GUM+LOCUST BEAN GUM (XG) 
The concentrations are given for preparing 45 g of gel. 225 mg of xanthan 
gum is dissolved in 15 ml of distilled water at approximately 70.degree. 
C. The same procedure is followed with 225 mg of locust bean gum instead 
of the xanthan gum. When the two solutions are at about 45.degree. C., 7.5 
ml of suspension is stirred into each. The mixture of suspension+carob 
seeds is then poured into the mixture of suspension+xanthan gum. The 
preparation is cooled to give a gel. The gel is either dried in air until 
dehydrated, or as with the AlG gel, 40% of its weight in silica is added 
to it. The mixture is dried at ambient temperature until the content of 
residual water is 125 g per 100 g of silica. 
3.2 INOCULA WITH CRUSHED NODULE INCLUSIONS 
100 ml of freshly picked Alnus glutinosa nodules are washed and then ground 
in a mixer, to give approximately 300 ml of a homogeneous suspension, 
which will be used to prepare gels based on alginate or xanthan gum with 
crushed nodule inclusions. The gels are air dried, then reduced to powder 
form. 
INOCULA BASED ON ALGINATE (AlG) 
For the preparation of 200 g of gel: 2 g of alginate is dissolved in 160 ml 
of suspension. 40 ml of a 6 g/l solution of CaSO.sub.4 .multidot.2H.sub.2 
O is added to form a gel. Part of the gel is dried to total dehydration at 
ambient temperature; another part has 40% of its own weight of silica 
added to it, and is then dried until the content of residual water is 125 
g per 100 g of silica. 
INOCULA BASED ON XANTHAN GUM - LOCUST BEAN GUM 
For the preparation of 360 g of gel: 1.8 g of xanthan gum is dissolved in 
120 ml of water at 70.degree. C., for 20 minutes with agitation, then the 
temperature is returned to about 45.degree. C. The same procedure is 
carried out with 1.8 g of locust bean gum instead of the xanthan gum. When 
the two solutions are at about 45.degree. C., 60 ml of the suspension of 
crushed nodules is added to each. The preparation is cooled to give a gel. 
Part of this is dehydrated, and the other part has silica added to it as 
with the alginate gel. 
4. RESULTS 
4a. TEST IN "POUCHES" 
The survival of the microorganism after inclusion, drying and storage at 
ambient temperature (20.degree.-25.degree. C.) has been checked on 
plantlets of Alnus glutinosa by the "Pouches" method described above. The 
plantlets arranged in the bag are developed for 10 days under controlled 
conditions, then they are inoculated by depositing the inoculum to be 
studied below the end of the main root. If inoculation is effective and 
the microorganism live, swellings will appear 10 to 20 days after 
inoculation, then small nodules on the root at the place where the 
inoculum was deposited. The results obtained with inocula based on AlG or 
XG gel+silica which have been stored for 10 to 20 days at ambient 
temperature are set out in Table I below. 
TABLE I 
______________________________________ 
Influence of type of inoculum on 
nodulation of Alnus glutinosa 
Preserva- 
Quantity of 
No. of nodulated 
tion inoculum per 
plants/total no. 
Inoculum (days) (4) 
plant of plants 
______________________________________ 
Not inoculated 
-- 0 0/18 
Suspension of 
0 0.25 ml 5/6 
crushed nodules.sup.(1) 
Crushed nodules 
20 about 5 mg 4/4 
included in dry 
XG.sup.(2) 
ARbNN4b 0 0.25 ml 1/5 
Liquid culture.sup.(3) 
included in XG + 
70 5 mg 6/9 
silica.sup.(5) 
included in AlG + 
70 5 mg 6/9 
silica 
AgN1ag 0 0.25 ml 6/6 
liquid culture.sup.(3) 
included in XG + 
20 5 mg 5/6 
silica.sup.(5) 
______________________________________ 
.sup.(1) 150 mg fresh nodules/5 ml H.sub.2 O 
.sup.(2) drying in air H = 12% 
.sup.(3) culture used as inclusion 
.sup.(4) preservation of inoculum at ambient temperature (20 to 25.degree 
C.) 
.sup.(5) precipitated silica - BET surface area = 200 m.sup.2 /g - CTAB = 
80 m.sup.2 /g. BET surface: determined in accordance with the 
BrunauerEmmett-Teller method, described in Journal of the American 
Chemical Society 60, p. 309 (1938). CTAB surface: outer surface by 
absorption of cetyl trimethyl ammonium bromide at pH 9, in accordance wit 
the method described by Jay, Janzen and G. Kraus in Rubber Chemistry and 
Technology 44, p. 1287-1296 (1971). 
It will be seen that there is approximately 100% nodulation in the cases 
where the inocula are prepared from included microorganisms+silica, 
although the quantity of culture used in 5 mg is 50 to 100 times less than 
that used in 0.25 ml of liquid inoculum. 
4. TESTS ON VERMICULITE 
The infectivity and effectiveness of the microorganism (stock ArbNN4b) 
after inclusion and storage at ambient temperature (20.degree. to 
25.degree. C.) have been tested with Alnus glutinosa developed in test 
tubes containing vermiculite+nutrient medium without any nitrogen (Crone 
solution). The results obtained are given in Table II. 
TABLE II 
__________________________________________________________________________ 
Influence of type of inoculum on 
nodulation and growth of 
Alnus glutinosa (age of plantlets 50 days) 
Average 
Weight in mg 
height 
of green 
of 
Preservation 
No. Nodules/ 
aerial part/ 
Plants 
Appearance 
Inoculum 
(days) (2) 
6 Plants 
6 Plants 
mm of Plants 
__________________________________________________________________________ 
Not -- 0 182 31 chlorosis 
inoculated 0 195 42 chlorosis 
liquid 
0 9 331 50 green 
culture 
Included 
15 30 468 56 green 
in XG + 
silica.sup.(1) 
Included 
30 36 515 59 green 
in XG + 
silica.sup.(1) 
Included 
30 32 444 62 green 
in AlG + 
silica 
__________________________________________________________________________ 
.sup.(1) Culture used as inclusion. 
.sup.(2) Preservation of inoculum at ambient temperature (20-25.degree. 
C.) 
These results show a certain novelty in this field, since this is the first 
time that anyone has succeeded in preparing inocula which included 
endophyte (pure culture of Frankia or suspension of crushed nodules), with 
the following advantages: 
(1) inocula in powder form, easy to handle; 
(2) possible use in very small volumes; 
(3) microorganisms included retain their infectivity when stored at ambient 
temperature; and 
(4) no problem of contamination by external agents. 
The process of including crushed nodules of Casuarina in an air-dried 
alginate gel has been used, in particular, for inoculating 600,000 
Casuarina stock in Senegal, as part of a project for immobilizing maritime 
and continental dunes. 
EXAMPLE 2: Mycorrhizae 
1.1: STOCK USED 
Pisolithus tinctorius (Marx) 
1.2: CULTIVATION OF MICROORGANISM IN LIQUID MEDIUM 
As a general rule the fungus is maintained and cultivated on a Marx medium 
as modified by Melin-Norkrans (MMN)*. Under these conditions fermentation 
takes from 6 to 8 weeks. 
*MMN (g/l): CaCl.sub.2, 0.05; NaCl, 0.025; K.sub.2 HPO.sub.4, 0.5; 
(NH.sub.4).sub.2 HPO.sub.4, 0.25; MgSO.sub.4 .multidot.7H.sub.2 O, 0.15; 
Ferric Citrate, 1 ml (1% Fe citrate); thiamine, 100 .mu.m; malt extract, 
3.0; saccharose, 10.0; H.sub.2 O to make 1000 ml (pH=5.5). 
One of the cultivating sequences adopted for Pisolithus is given below: 
##STR1## 
INOCULUM CULTURE I 
receptacle: 300 ml Erlenmeyer flask plugged with polyurethane cotton, 
filled with 70 ml of culture medium. 
PEG culture medium (g/l): peptone, 10; yeast extract, 5; glucose, 10; 
gelose, 1; pH=approximately 7; sterilization; 25 minutes at 120.degree. 
C.; pH after sterilization=approximately 6.6; 
seeded from a culture on gelose (fragments of gelose+mycelium); 
conditions: incubated 10 to 15 days on agitating table rotating at 100-140 
rpm, tray 25 mm off center, temperature 28.degree. C. 
INOCULUM CULTURE II 
The cultivating conditions are the same as above, but seeding takes place 
from culture I in quantities of 5 to 10%, and the incubating time is 
reduced to one week. 
______________________________________ 
Productive culture in 2 liter fermenter 
(Biolafitte) 
______________________________________ 
filling: 1000 ml 
medium: PEG described above 
seeding: 1 to 10% 
conditions: agitation with turbine turning at 
250-300 rpm, aeration 20 1/hour, 
temperature 25 to 30.degree. C. 
______________________________________ 
RESULTS 
After 8 to 13 days of cultivation in a 2 liter fermenter, a dense, 
homogeneous culture is obtained, thereby avoiding any subsequent crushing 
of the mycelium before the inoculum is prepared. The quantity of biomass 
(dry weight at 105.degree. C.) obtained per liter of medium is in the 
vicinity of 2.5 g/l. 
1.3 PREATION OF INOCULA WITH INCLUDED MICROORGANISMS 
Since ectomycorrhizian fungi do not have any form of resistance when 
cultivated in pure culture, it is necessary to retain a certain content of 
residual water when the inoculum is being prepared, in order to insure the 
survival of the mycelium. This type of inoculum has been made either by 
adding silicas to the gel following the inclusion of the fungus in a gel 
based on polysaccharides, or by mixing the mycelium suspension with 
alginate and letting the mixture fall drop by drop into a concentrated 
solution of CaCl.sub.2, on the principle described by Hackel in 1977 as 
applied to the immobilization of Rhizobium (French patent application 
79.28956 (1979)). 
INOCULUM IN POWDER FORM 
The culture developed in a 2 liter fermenter is filtered or centrifuged, 
washed and suspended in physiological salt solution. The suspension is 
used to prepare gels based on alginate or xanthan gum, by the methods 
described previously for Frankia. 
The gels then have synthetic or natural silica added to them and are mixed, 
then dried, until the residual water content is from 70 to 250%, thus 
giving moist or partially dehydrated powders which are kept in sealed 
bottles. 
INOCULUM IN FORM OF ALGINATE PELLETS 
From 1 to 2 g/l of alginate is dissolved in the mycelium suspension, then 
the mixture is added drop by drop to a concentrated 170 g/l solution of 
CaCl.sub.2. The pellets formed are rapidly removed, then washed in tap 
water and kept in sealed bottles. 
The effective life of the fungus (Pisolithus tinctorius) in the 
preparation, particularly in the case of alginate pellets, is over six 
months at ambient temperature. The test used to assess survival is based 
on the principle of colorimetric determination of respirometric activity 
by reducing Nitro Blue Tetrazolium to formazan, a compound colored blue. 
The infectivity and effectiveness of the inocula is tested on Pinus 
caribaea or Pinus pinaster when the pellets have been redissolved by means 
of a decomplexing agent, such as phosphates, citrate, lactate, etc. 
These tests illustrate the progress made in the preparation of inocula with 
actomycorrhizae. The cultivating of currently used mycorrhizian fungi in a 
liquid medium is improved with respect to production of homogeneous 
biomass and reduction of fermentation time by four to six weeks. Moreover, 
the procedure allows for the preparation of inocula which are easy to use 
and enable the microorganism to survive for several months. 
The method of preparing inocula with Frankia and ectomycorrhizae may also, 
for example, be applied to the inclusion of endomycorrhizae with clusters 
developed in pure culture, and to the inclusion of crushed roots 
mycorrhiza (treated by endomycorrhizae with vesicles and arbuscles). 
Inocula prepared by including roots which have been infected then crushed 
(mycelium+spores) in XG or AlG gels with silica added retain their 
infectivity: the endomycorrhizian fungus with vesicles and arbuscles is 
present on the roots of the test plant inoculated by this process. 
The terms and expressions which have been employed are used as terms of 
description and not of limitation, and there is no intention in the use of 
such terms and expressions of excluding any equivalents of the features 
shown and described or portions thereof, but it is recognized that various 
modifications are possible within the scope of the invention claimed.