CA1266842A - Process for culturing fungi in vitro from vesicular- arbuscular mycorrhizae - Google Patents
Process for culturing fungi in vitro from vesicular- arbuscular mycorrhizaeInfo
- Publication number
- CA1266842A CA1266842A CA000487287A CA487287A CA1266842A CA 1266842 A CA1266842 A CA 1266842A CA 000487287 A CA000487287 A CA 000487287A CA 487287 A CA487287 A CA 487287A CA 1266842 A CA1266842 A CA 1266842A
- Authority
- CA
- Canada
- Prior art keywords
- mycorrhizae
- fungi
- vesicular
- vitro
- arbuscular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/10—Mycorrhiza; Mycorrhizal associations
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Mushroom Cultivation (AREA)
Abstract
PROCESS FOR CULTURING FUNGI IN VITRO FROM
VESICULAR-ARBUSCULAR MY CORRHIZAE
Inventors: Désiré-Georges STRULLU
Corinne ROMAND
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) ABSTRACT
The invention relates to a process for culturing in vitro in an axenic condition fungi of the type Endogonaceae, especially of the genus Glomus, which process consists in removing vesicular-arbuscular mycorrhizae, cleaning them and sterilizing them, and then placing the mycorrhizae in culture on a sterile agar nutrient medium provided with mineral elements, vitamins, especially of the 3 group, and sugar. The invention also concerns the pro-duction of mycelium and mycorrhizae from mycorrhizae obtained under sterile conditions. The product of the invention is useful for fertilizing products.
VESICULAR-ARBUSCULAR MY CORRHIZAE
Inventors: Désiré-Georges STRULLU
Corinne ROMAND
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) ABSTRACT
The invention relates to a process for culturing in vitro in an axenic condition fungi of the type Endogonaceae, especially of the genus Glomus, which process consists in removing vesicular-arbuscular mycorrhizae, cleaning them and sterilizing them, and then placing the mycorrhizae in culture on a sterile agar nutrient medium provided with mineral elements, vitamins, especially of the 3 group, and sugar. The invention also concerns the pro-duction of mycelium and mycorrhizae from mycorrhizae obtained under sterile conditions. The product of the invention is useful for fertilizing products.
Description
~26684Z
The present invention relates to a process for obtaining, in an axenic condition in vitro, fungi from vesicular-arbuscular mycorrhizae.
The invention also relates to the fertilizing pro-ducts involving the application of the fungi obtainedaccording to th;s or a similar process.
3efore the main provisions of the invention are dealt ~;th, it is appropriate to recall the difficult;es encountered ~hen attempts have been made to culture such 1û fungi in the pure state.
Scientific ~ork performed in a closely related f;eld has already enabled inocula to be produced in v;tro.
All the ;nocula of ves;cular-arbuscular mycorrh;zal fungi currently produced i _ itro have been obtained from spores of these fungi.
The mycel;a of ves;cular-arbuscular mycorrh;zal fungi, aris;ng from spores germinated under ster;le con-d;t;ons, are at present only cultured in the presence of a host plant or a portion of the latter. The culturing of these mycelia ;n v;tro on an inorganic and organic medium, ~ithout a host plant, has not been performed.
The processes of culture in the presence of a host plant, performed ;n v;tro, are difficult to carry out. In effect, ;t ;s necessary to culture, ;n one and Z5 the same nutrient medium, tvo organisms having different requirements for achieving optimal development. Several processes also ex;st for produc;ng inoculum of ves;cular-arbuscular endomycorrh;zal fung; from spores or from mycorrh;zed roots, under non-ster;le conditions. All .
these non-axenic processes have the d;sadvantage of con-stituting a source of introduction of pathogenic micro-organisms.
The object of the present invention is conse-quently to provide, first, a culture process for obtainingin v;tro, ;n a ster;le med;um, fungi forming vesicular-arbuscular mycorrhizae, for the purpose of promoting the mineral nutr;t;on of plants of agricultural value such as wheat, ma;ze, pea, potato, tomato, soya, clover, onion, strawberry and asparagus. Spec;es of hort;cultural value are also ;nvolved ~tulip, rose) as well as valuable forest tree species (ash, wild cherry, tul;p tree).
Another object of the ;nvent;on ;s to provide vesicular-arbuscular endomycorrhizal fungi which do not possess host specific-ity a~d which make ;t possible to mycorrhize a very large number of plant species, which are consequently not limited to those mentioned above by way of example.
~ To this end, the invention relates to a process ;~ ~ 20 for culturing ;n vitro, in an axenic condition, fung; of the type Endogonaceae, especialLy of the genus Glomus, which process consists in removing ves;cular-arbuscular ~mycorrhizae, cleaning them, ;n particular by ultra-sound, and sterilizing them, for example by immersion in disin-25~ fectant solutions, and then placing them on a ster;le : ~ ~
~ nutrient~medium provided with mineral elements, vitamins, ." .
especially of the ~ group, and sugar; as a result of which, surprisingly, growth of the mycelium is observed, in particular from the ends of the mycorrhiza.
- , : - :
: ~ : , '-':. ' ' ~ . , ' - ' ~2636842 Although the cleaning of the vesicular-arbuscular mycorrhizae is preferably performed w;th ultrasound, it is poss;ble to use other clean;ng techn;ques.
S;m;larly, the ster;l;zat;on can employ d;s;nfec-tant solut;ons such as ant;b;ot;cs or calcium hypochlor-ite, alone or in success;on, but other types of sterili-zat;on can also be used.
Of course, ;f the ves;cular-arbuscular mycorrh;zae are sterile at the start, there is no need to d;sinfect them.
The process enables mycelia and mycorrhizae to be obtained under sterile conditions, which entitles them to be exported without their be;ng hygiene p~oblems.
Finally, the vitamins of the B group which can be employed can be, in particular: calc;um panthothenate, biotin, n;cotinic acid, pyridoxine ~B6), and thiamine ~81).
Accord;ng to a characteristic of this process, an inoculum is produced which can be used for the mycorrh;za-tion of seedlings in a sterile or non-ster;le culture med;um.
The mycorrhizae obtained can themselves be used to produce mycelium or to cause mycorrhization.
Other characteristics and advantages of the present process will also emerge from the detailed description below of an embodiment of the present process, and from the drawings whicb Figures 1 and 2 illustrate, by way of example, a parent root which has produced the mycelium in sterile medium.
According to the invention, roots of strawberry plants are removed from host plantu whlch have risen In , ~266842 ~,~
natural or mycorrhized conditions in pots. The mycorrhi-zation is ver;fied by conventional sta;n techniques.
The mycorrhizae are vibrated with ultrasound for approximately 15 minutes, and then fragmented ;nto 0.3-5 to 1-cm segments. These segments are then d;sinfected by passage into 95 alcohol solution for 2 to 10 m;nutes, and r;nsed w;th ster;le d;st;lled water. Under a ster;le hood, the spec;mens are d;s;nfected by passage ;nto 6X
strength calc;um hypochlor;te solut;on for 1 to 10 m;nutes, 10 and then r;nsed three t;mes w;th ster;le d;st;lled water.
D;sinfect;on is f;nally performed by passage ;nto an ant;-b;ot;c solut;on (200 ppm streptomyc;n + 2X chloramine T) for 5 to 20 m;nutes, followed by r;ns;ng w;th ster;le d;st;lled water.
15 The root fragments are cultured ;n the dark at 25C, ;n particular ;n Petr; d;shes conta;n;ng the fol-low;ng med;um:
a) M;neral elements ~Hepper, 1981) Major elements mg l 20 . KN03 : 303 . Ca (N03)2. 4H20 : 2,040 . MgS04. 7H20 : 368 . KH2P04 44 . FeNaEDTA : 65 Trace elements mg l . MnS04. 4H20 2.23 . CuS04. 5H20 0.24 . ZnS04. 7H20 0.29 H3~03 1.86 iZ6684;2 . (NH4)6Mo7024~ 4H20 b~ Vitamins of the 8 group mg l 1 . Calc;um panthothenate : 1 . Biotin : 0.01 5 . Nicot;n;c ac;d . Pyridoxine (B6) : 1 . Th;am;ne (81) . V;tamin B12 : 0.5 c) Sucrose 15 9 l~
10 d) Agar 7 9 l 1 e) pH before autoclav;ng 7.
After a few days, the growth of mycel;ohyphae is observed from root sect;ons. Somet;mes, hyphae also emerge from the roots by break;ng through the epidermis.
The appearance of the cultures ;s shown ;n F;gures 1 and
The present invention relates to a process for obtaining, in an axenic condition in vitro, fungi from vesicular-arbuscular mycorrhizae.
The invention also relates to the fertilizing pro-ducts involving the application of the fungi obtainedaccording to th;s or a similar process.
3efore the main provisions of the invention are dealt ~;th, it is appropriate to recall the difficult;es encountered ~hen attempts have been made to culture such 1û fungi in the pure state.
Scientific ~ork performed in a closely related f;eld has already enabled inocula to be produced in v;tro.
All the ;nocula of ves;cular-arbuscular mycorrh;zal fungi currently produced i _ itro have been obtained from spores of these fungi.
The mycel;a of ves;cular-arbuscular mycorrh;zal fungi, aris;ng from spores germinated under ster;le con-d;t;ons, are at present only cultured in the presence of a host plant or a portion of the latter. The culturing of these mycelia ;n v;tro on an inorganic and organic medium, ~ithout a host plant, has not been performed.
The processes of culture in the presence of a host plant, performed ;n v;tro, are difficult to carry out. In effect, ;t ;s necessary to culture, ;n one and Z5 the same nutrient medium, tvo organisms having different requirements for achieving optimal development. Several processes also ex;st for produc;ng inoculum of ves;cular-arbuscular endomycorrh;zal fung; from spores or from mycorrh;zed roots, under non-ster;le conditions. All .
these non-axenic processes have the d;sadvantage of con-stituting a source of introduction of pathogenic micro-organisms.
The object of the present invention is conse-quently to provide, first, a culture process for obtainingin v;tro, ;n a ster;le med;um, fungi forming vesicular-arbuscular mycorrhizae, for the purpose of promoting the mineral nutr;t;on of plants of agricultural value such as wheat, ma;ze, pea, potato, tomato, soya, clover, onion, strawberry and asparagus. Spec;es of hort;cultural value are also ;nvolved ~tulip, rose) as well as valuable forest tree species (ash, wild cherry, tul;p tree).
Another object of the ;nvent;on ;s to provide vesicular-arbuscular endomycorrhizal fungi which do not possess host specific-ity a~d which make ;t possible to mycorrhize a very large number of plant species, which are consequently not limited to those mentioned above by way of example.
~ To this end, the invention relates to a process ;~ ~ 20 for culturing ;n vitro, in an axenic condition, fung; of the type Endogonaceae, especialLy of the genus Glomus, which process consists in removing ves;cular-arbuscular ~mycorrhizae, cleaning them, ;n particular by ultra-sound, and sterilizing them, for example by immersion in disin-25~ fectant solutions, and then placing them on a ster;le : ~ ~
~ nutrient~medium provided with mineral elements, vitamins, ." .
especially of the ~ group, and sugar; as a result of which, surprisingly, growth of the mycelium is observed, in particular from the ends of the mycorrhiza.
- , : - :
: ~ : , '-':. ' ' ~ . , ' - ' ~2636842 Although the cleaning of the vesicular-arbuscular mycorrhizae is preferably performed w;th ultrasound, it is poss;ble to use other clean;ng techn;ques.
S;m;larly, the ster;l;zat;on can employ d;s;nfec-tant solut;ons such as ant;b;ot;cs or calcium hypochlor-ite, alone or in success;on, but other types of sterili-zat;on can also be used.
Of course, ;f the ves;cular-arbuscular mycorrh;zae are sterile at the start, there is no need to d;sinfect them.
The process enables mycelia and mycorrhizae to be obtained under sterile conditions, which entitles them to be exported without their be;ng hygiene p~oblems.
Finally, the vitamins of the B group which can be employed can be, in particular: calc;um panthothenate, biotin, n;cotinic acid, pyridoxine ~B6), and thiamine ~81).
Accord;ng to a characteristic of this process, an inoculum is produced which can be used for the mycorrh;za-tion of seedlings in a sterile or non-ster;le culture med;um.
The mycorrhizae obtained can themselves be used to produce mycelium or to cause mycorrhization.
Other characteristics and advantages of the present process will also emerge from the detailed description below of an embodiment of the present process, and from the drawings whicb Figures 1 and 2 illustrate, by way of example, a parent root which has produced the mycelium in sterile medium.
According to the invention, roots of strawberry plants are removed from host plantu whlch have risen In , ~266842 ~,~
natural or mycorrhized conditions in pots. The mycorrhi-zation is ver;fied by conventional sta;n techniques.
The mycorrhizae are vibrated with ultrasound for approximately 15 minutes, and then fragmented ;nto 0.3-5 to 1-cm segments. These segments are then d;sinfected by passage into 95 alcohol solution for 2 to 10 m;nutes, and r;nsed w;th ster;le d;st;lled water. Under a ster;le hood, the spec;mens are d;s;nfected by passage ;nto 6X
strength calc;um hypochlor;te solut;on for 1 to 10 m;nutes, 10 and then r;nsed three t;mes w;th ster;le d;st;lled water.
D;sinfect;on is f;nally performed by passage ;nto an ant;-b;ot;c solut;on (200 ppm streptomyc;n + 2X chloramine T) for 5 to 20 m;nutes, followed by r;ns;ng w;th ster;le d;st;lled water.
15 The root fragments are cultured ;n the dark at 25C, ;n particular ;n Petr; d;shes conta;n;ng the fol-low;ng med;um:
a) M;neral elements ~Hepper, 1981) Major elements mg l 20 . KN03 : 303 . Ca (N03)2. 4H20 : 2,040 . MgS04. 7H20 : 368 . KH2P04 44 . FeNaEDTA : 65 Trace elements mg l . MnS04. 4H20 2.23 . CuS04. 5H20 0.24 . ZnS04. 7H20 0.29 H3~03 1.86 iZ6684;2 . (NH4)6Mo7024~ 4H20 b~ Vitamins of the 8 group mg l 1 . Calc;um panthothenate : 1 . Biotin : 0.01 5 . Nicot;n;c ac;d . Pyridoxine (B6) : 1 . Th;am;ne (81) . V;tamin B12 : 0.5 c) Sucrose 15 9 l~
10 d) Agar 7 9 l 1 e) pH before autoclav;ng 7.
After a few days, the growth of mycel;ohyphae is observed from root sect;ons. Somet;mes, hyphae also emerge from the roots by break;ng through the epidermis.
The appearance of the cultures ;s shown ;n F;gures 1 and
2. The f;laments form a loosely packed network, w;th l;ttle branch;ng, of slender hyphae.
Equ;valen.t results are observed follow;ng the process of Example 1 but start;ng w;th tul;p tree or alfalfa roots.
Equivalent results are observed following the process of Example 1, but starting with roots mycorrh;zed under sterile cond;t;ons.
In this example, legum;nous plants such as clover can advantageously be used. ~t ;s obv;ous that ;t ;s unnecessary to d;s;nfect these roots wh;ch have been mycorrh;zed under ster;le cond;t;ons.
,. - ,~ '
Equ;valen.t results are observed follow;ng the process of Example 1 but start;ng w;th tul;p tree or alfalfa roots.
Equivalent results are observed following the process of Example 1, but starting with roots mycorrh;zed under sterile cond;t;ons.
In this example, legum;nous plants such as clover can advantageously be used. ~t ;s obv;ous that ;t ;s unnecessary to d;s;nfect these roots wh;ch have been mycorrh;zed under ster;le cond;t;ons.
,. - ,~ '
Claims (9)
1. A process for culturing in vitro in an axenic condition fungi of the type Endogonaceae, which process consists in isolating vesicular-arbuscular mycorrhizae, cleaning them and sterilizing them, and placing the mycorrhizae in culture on a sterile agar nutrient medium provided with mineral elements, vitamins, and sugar.
2. The process as claimed in claim 1, wherein the fungi of the type Endogonaceae is of the genus Glomus
3. The process as claimed in claim 1, wherein the sterilization is performed by immersing the mycorrhizae in at least one disinfectant solution.
4. The process as claimed in claim 3, wherein the disinfectant solution contains an antibiotic.
5. The process as claimed in claim 3 or claim 4, wherein the disinfectant solution is calcium hypochlorite solution.
6. The process as claimed in claim 1, 2 or 3, wherein the cleaning is performed by an ultrasonic treatment.
7. The process as claimed in claim 1, 2 or 3, wherein the vitamins of the B group present in the nutrient medium are chosen from calcium panthothenate, biotin, nicotinic acid, pyridoxine, thiamine and vitamin B12.
8. The process as claimed in claim 1, 2 or 3, wherein fungal structures of intra-root origin are used.
9. A process for mycorrhization of roots of plants of agricultural value, which comprises applying thereto the mycelia or mycorrhizae obtained by the process of claim 1, 2 or 3.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8411725 | 1984-07-24 | ||
| FR8411725A FR2568094B1 (en) | 1984-07-24 | 1984-07-24 | PROCESS FOR THE IN VITRO CULTURE OF MUSHROOMS FROM VESICULAR AND ARBUSCULATED MYCORHIZES |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1266842A true CA1266842A (en) | 1990-03-20 |
Family
ID=9306431
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000487287A Expired - Fee Related CA1266842A (en) | 1984-07-24 | 1985-07-23 | Process for culturing fungi in vitro from vesicular- arbuscular mycorrhizae |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0172085B1 (en) |
| AT (1) | ATE49846T1 (en) |
| CA (1) | CA1266842A (en) |
| DE (1) | DE3575655D1 (en) |
| FR (1) | FR2568094B1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU5940590A (en) * | 1989-07-14 | 1991-02-22 | Sean Morrison | Production of mycorrhizal inoculum by static culture hydroponics |
| US5002603A (en) * | 1989-12-04 | 1991-03-26 | Board Of Trustees Operating Michigan State University | Method and compositions for stimulating vesicular-arbuscular mycorrhizal fungi |
| FR2856553B1 (en) * | 2003-06-24 | 2007-06-01 | Univ Angers | PROCESS FOR IN VITRO PRODUCTION OF MYCORRHIZAL FUNGI, MYCOCAL AND MYCORHIZED BIOLOGICAL SUPPORT OBTAINED |
| ES2268984B1 (en) * | 2005-07-29 | 2008-03-16 | Consejo Superior Investig. Cientificas | ASEPTIC INOCULATING MICORRIZATION AND APPLICATION PROCEDURES IN CONDITIONS IN VITRO AND EX VITRO. |
| LU92274B1 (en) | 2013-08-30 | 2015-03-02 | Symplanta Gmbh & Co Kg | System and methods for continuous propagation and mass production of arbuscular mycorrhizal fungi |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2528865A1 (en) * | 1982-06-21 | 1983-12-23 | Rhone Poulenc Sa | PROCESS FOR OBTAINING IN VITRO ENDOMYCORHIZIC FUNGI WITH VESICLES AND ARBUSCLES |
-
1984
- 1984-07-24 FR FR8411725A patent/FR2568094B1/en not_active Expired
-
1985
- 1985-07-22 AT AT85401507T patent/ATE49846T1/en not_active IP Right Cessation
- 1985-07-22 EP EP85401507A patent/EP0172085B1/en not_active Expired - Lifetime
- 1985-07-22 DE DE8585401507T patent/DE3575655D1/en not_active Expired - Fee Related
- 1985-07-23 CA CA000487287A patent/CA1266842A/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| EP0172085A1 (en) | 1986-02-19 |
| FR2568094B1 (en) | 1987-05-29 |
| DE3575655D1 (en) | 1990-03-08 |
| ATE49846T1 (en) | 1990-02-15 |
| FR2568094A1 (en) | 1986-01-31 |
| EP0172085B1 (en) | 1990-01-31 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |