Note: Descriptions are shown in the official language in which they were submitted.
5~7~ -
The invention concerns a process for the production
of conidia of entomophagic fungi by superficial (surface)
cultivation on a nutrient medium containing assimilable
carbon and nitrogen sources and nutritive mineral salts.
It is known that spores of entomophagic fungi
possess a marked insecticidal activity against agricultural
pests. The interest in entomophagic fungi has been con-
tinuously growing, especially in connection with the
requirements of modern environment protection methods (C.M.
Ignoffo, Environm. Lett. 8, 23, 1975; N.V. Lappa et al.,
Zashtchita rastenii 24, 1977). The chemical pesticides in
present usage possess relatively broad spectra of action and
consequently hamper the natural equilibrium; the pests rapidly
develop resistance to the pesticides, whose residues con-
taminate agricultural products. Successful applications of
entomophagic fungi have documented the advantages of certain
species, in particular from the class Deuteromycetes. One
of such species is, for example, the fungus Beauveria bassiana
(Bals.) Vuill~ (J. Kral. S. Neubauer, Zool. ent. Listy 1953,
241; V. M. Goral, N. V. Lappa, Zachist, roslin 20, 51, 1974;
P. Ferron, Entomophaga 19, 103, 1974; A. Samsinakova, A.
Germakova, Sbornik Cs. akad. zem. ved, Rostlinna vyroba 6,
163, 1960). The endeavour for a large-scale realization
has encountered, up to the present, the difficulties con-
nected with technologically acceptable procedures for obtain-
ing the great masses of spores necessary for the production
of an efficient preparation under economically favourable
conditions ( ~cCoy et al., J. Invert. Pathol. 19, 370, 1972;
Entomophage 20, 229, 1975; O. A. Alioshine et al., Mikrobiol.
Promyshlennost 5, 18, 1976; J. Leopold et al., Zbl. Bakt.
Abt. 11, 128, 31, 1973). Submersion cultivation of spores
in a synthetic nutrient medium on a production scale is
--1-- ~!
~i
feasible, but it was found that under such conditions there
formed only the blastoarthrospores, which in the course of
the finalization and preservation operations of the product
considerably lost their virulence. In consequence of this
the method had to be abandoned, and sumbersed spores serve
solely, especially to save time, for inoculation of nutrient
media for stationary cultivation of aerial spores. The
aerial spores, produced by abstriction from the tips of the
conidiophores, evsn after drying keep their full activity
for long periods of time (A. Samsinakova, J. Invert. Pathol.
8, 395, 1966; N. A. Telenga, V. M. Goral, Biol. met. bordy
1967, 171).
For the production of aerial conidia of the fungus
Beauveria bassiana there have been used various natural
solid substrates, such as potatoes, sugar beet bulbs, cereals,
and others, and the cultivation yield has been often assessed
merely in terms of the dry mass of the produced mycelium,
without any statement of the number of spores or any nearer
specification of the final biological preparation. According
to the first quantitative data, an infectious product was
successfully grown on potato paste in Petri dishes, with a
content of 1.5 x 109 conidia per each gram of dry biomassO
Each 100-cm2 cultivation area yielded 14 x 109 conidia.
For treatment of each hectare (10 000 m2) of potato field,
4 kg of this preparation were needed (J. Kral, S. Neubauer,
Zool. Listy V, 178, 1956). Similar results were achieved
on a molasses-containing medium, sugar-beet decoction, and
solid agar media (N. A. Telenga, V. M. Goral, 1. c.; A. A.
Yevlakhova, L. G. Tarasov, Biol. Method of Plant Pest Control,
Riga 1968, 131; E. V. Kononova, Proc. First Joint USSUSSR
Conference, Kiev 1979, 173). On the basis of these experiences,
the production was started of a commercial preparation with
27~
the s-tandard titer 6 x 109 spores per gram (C. M. Ignoffo
et al., J. Econ. Ent. 72, 562, 1979). Nevertheless, for
an effective and economic application in the conditions of
agricultural large~scale production the possibilities of all
methods applied up to the present have been strongly limited,
and the obtained products, too expensive.
It would be advantageous to avoid the above draw- !
backs for obtaining the conidia of entomophagic fungi, in f
; particular of the fungus Beauveria bassiana (Bals.) Vuill.
The present invention provides a process for the
production of conidia of entomophagic funyi by surface cul-
tivation on a nutrient medium containing assimilable carbon
and nitrogen sources and nutritive mineral salts, characterized
in that the cultivation is conducted on a liquid nutrient
medium with a pH value of 4.0 to 6.0 containing, as the as-
similable carbon source, a nutrient whose assimilation rate
is lower than that of glucose or sucrose in a mass concentra-
tion of 0.5 % to 3.0 ~ and as the assimilable nitrogen source,
a natural organic nitrogenous substance, in a mass concen-
tration of 0.8 ~ to 2.0 %.
In particular the present invention provides a
- process for the production of conidia of the fun~us Beauveria
bassiana (Bals.)~uill. by surface cultivation on a nutrient medium contain-
in~ assimilable carbon and nitrogen sources and nutritive
mineral salts, characteri~ed in that the cultivation is con-
ducted on a liquid nutrient medium with a pH value of 4.0 to
6.0, containing, as the assimilable carbon source, a nutrient
whose assimilation rate is lower than that of glucose or
sucrose in a mass concentration of 0.5 % to 3.0 ~ and as the
assimilable nitrogen source, a natural organic nitrogenous
substance, in a mass concentration of 0.8 % to 2.0 ~.
As indicated above our procedure is es~entially
-3-
based on cultivation upon a medium with p~ 4.0 to 6.0,
preferably 5.0, containing an assimilable carbon source
; with an assimilation rate slower than that of glucose or
sucrose, for example, alcoholic sugars, preferably sorbitol
and/or mannitol, in mass concentrations of 0.5 % to 3.0 %,
preferably 1 %~ and a na-tural, organic source of assimilable
nitrogen, for example, corn steep, casein hydrolysate, or
peptone, in mass concentrations of 0.8 ~ to 2.0 ~.
Another advantage of our method of superficial
(surface) cultivation on a liquid medium consists in its
feasibility upon large areas, following the procedure for
microorganism cultivation described in the Czechoslovak
Patent No. 172 557; according to this patent the cultivation
is carried out in a sealable, sterile, inflated bag of a
transparent plastic foil, preferably polyethylene foil,
provided with at least one inlet for the introduction of
liquid, gaseous, and/or solid (slurried) ingredients, serving
as nutrients and/or inoculum and/or air for the culture.
The cultivation bags are expediently incubated upon a horizon-
tal support plate provided with te~lperature regulation.
An especially valuable.feature of our invention isthat on our liquid medium we have succeeded in achieving
- a spore production, per area unit, by up to two decimal orders
higher in comparison with the yields achieved with the pre-
sently used solid, pasty, or powdery substrates, and have
obtained 1 x 1011 pure aerobic conidia per each 100-cm
surface area.
The spore production intensity is considerably
influenced by the choice of a suitable carbon source. Not
suitable have been proved the readily assimilable hexoses
(glucose or fructose), but also sucrose, maltose, and water-
soluble starch. This experience points to high activities
'
_~_
of the ~-D-fructofuranosidase, amylase, and maltase, which
make possible unlimited glycolysis and thus foster the fungal
mycelium growth at the cost of spore production. We achieved
the maximal spore production when using a slowly assimilable
carbon source, above all, sorbitol.
Out of industrially available nitrogen sources,
some natural organic mixtures have been proved best, such as
peptone, casein hydrolysate, or corn steep.
According to the present invention the prefered
spores are those of the species Beauveria bassiana. As
examples of spores of other entomophagic fungi which can be
obtained there can be cited the Paecilomyces, Verticillium,
and Culicinomyces species.
A great asset of our procedure consists in its
feasibility on a large scale, without complicated production
equipment, in polyethylene foil cultivation bags, even upon
areas of several square meters (Czechoslovak Patent No. 172
552). This procedure, in addition, makes possible a simple
separation of the highly conidia-rich biomass from the
remaining medium, which latter, after the conclusion of
incubation, is simply allowed to-drain off. The remaining
~iomass is then pressed, crushed, and dried by air stream.
Nearer details of our new procedure are evident
from the following example.
Example
For cultivation there was used the Beauveria
bassiana strain No. 30 from the mycoligical collection of
the Insect Pathology nepartment, Entomological Institute
of the Czechoslovak Academy of Sciences. The strain had
been isolated from an imago of the family Chrysomelidae,
and possesses the maximal efficacy against the Colorado
potato beetle (Leptinotarsa decemlineata Say). AFter 14-day
--5--
7~
cultivation on Sabouraud's agar at 27 C the conidia were
scraped off -the surface and mixed with sterile absorbent
charcoal. A preserve prepared in this way and stored at
7 C to 10 C keeps viable and virulent for several years.
The preserve served Eor inoculating a liquid
nutrient medium composed as follows (in mass per cent):
2.5 % glucose, 2.5 % soluble starch, 2.0 g6 corn steep, 0.2 %
CaC03, O.5 % NaCl, pE~ 5Ø The inoculated medium was cul-
tivated on a reciprocal shaker at 27 C for 72 hours. With
25 ml of this inoculum, 2 000 ml of previously sterilized and
cooled sporulation medium was inoculated and transferred
into a cultivation bag with a 0.1-m2 area (made according
to Czechoslovak Patent No. 172 552). The sporulation medium
contained 1 % sorbitol and 2.5 % peptone (Bactopepton Spofa);
pH 5Ø The polyethylene foil bags were incubated in the
horizontal position at 27 C for 12 clays. From day 3 onwards,
air was continuously blown over the qrowing, conidia-producing
mycelium at the rate of 400 ml/min. After separation and
drying of the biomass, 1 x 1013 serial conidia were obtained
20 Erom each bag.
