Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
2002-02-04
2003-07-15
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S256700, C435S256300, C435S256800
Reexamination Certificate
active
06593127
ABSTRACT:
FIELD OF THE PRESENT INVENTION
The present invention relates to a composition useful for early and profuse sporulation in fungi with about 100 fold increase in spore count within 48 hours of inoculation, said composition comprising molasses ranging between 5-20 g/L, Corn Steep Liquor (CSL) ranging between 10-25 g/L, Sodium chloride (NaCl) ranging between 5-15 g/L, calcium sulphate (CaSO
4
) ranging between 0.1-0.5 g/L, Potassium dihydrogen phosphate (KH
2
PO
4
) ranging between 0.001-0.01 g/L, Magnesium sulphate (MgSO
4
.7H
2
O) ranging between 0.001-0.01 g/L, Copper sulphate (CuSO
4
) ranging between 0.001-0.0050 g/L, Ferrous sulphate (FeSO
4
) ranging between 0.0009-0.005 g/L, an anti-foam agent, and optionally solidifying agent and a method thereof.
BACKGROUND AND PRIOR ART REFERENCES
Trichoderma species are used as efficient biocontrol agents against the soil-borne Wilt and Root-rot diseases of various crop plants. With the growing awareness of the harmful effects of many synthetic chemical pesticides on humans, non-target animals and plant species, and the environment as a whole, there has been a shift in attention to the research and development of more environment-friendly methods of pest and disease control.
Use of environmentally safe Disease Management Technologies based on Biopesticide and Biocontrol agents through natural enemies—parasitoids, predators and pathogens in integrated pest management programmes are ever increasing. Biocontrol agents like Trichoderma species and Gliocladium species are examples of some of the biocontrol agents used in the management of root diseases of chickpea, peas, groundnut, soybean spices and vegetables.
Root-rot diseases of crops in general and pulses in particular are a serious problem in rain-fed areas caused mostly by species of Fusarium and Rhizoctonia etc. Chemical control of these diseases requires a continuous use of fungicides, which is not economical and causes deleterious effects on the rhizosphere microflora and also have the residual problems.
WHO now has banned even some of the commonly used fungicides due to their severe toxicity. Under these circumstances the biocontrol agents can be used for the management of Root-rot diseases at comparatively cheaper cost with long term antagonistic effects against the pathogens because these biocontrol agents multiply and remain near the root zone of the plant giving protection to these plants throughout the growth period.
Investigations carried out world over have confirmed, that synthetic plant protection chemicals such as fungicides, insecticides and herbicides deteriorate soil fertility. Such deterioration occurs through disappearance of bio-diversity in soils. On account of this, several strains of soil microorganisms have been used which are able to suppress a wide range of disease causing microorganisms.
Jackson et al., (Enzyme-Microb. Technol.; (1991) 13, 6, 456-61) reported the culture media optimisation for production of the biological control agents
Gliocladium virens
G20
, Trichoderma pseudokoningii
IMI 322662, and
T. viride
IMI 322659 and IMI 322663. In glucose-alanine medium, the optimum dry wt./g carbon occurred with a C:N ratio of 15:1. Addition of 3.28-mg atoms iron/l to the medium increased biomass production of all isolates, but a concentration of 164-mg atoms/l was toxic to the Trichoderma species. Growth decreased in media lacking Mg, P, K or S, but the amount of the decrease differed between the four isolates. Sporulation in agar was reduced in the absence of Mg, P, K and N. Addition of biotin, p-amino -benzoic acid and thiamine-HCl increased biomass production slightly. The glucose-alanine basal medium supported better growth of all 4 isolates than a commercial molasses-yeast medium; conidia production was greater in the molasses-yeast medium. The pH of the glucose-alanine medium remained constant at 4.5, whereas the pH of the yeast-molasses medium (initially 5.5) increased to 8.0-8.6. Chlamydospores were produced by all isolates, but the numbers varied according to the culture conditions used.
Gervais and Sarrette (J.Fement.Bioeng. (1990, 69, 1, 46-50) reported the Emerson agar medium (18 g/l agar) containing sodium octanoate (1 g/l) as a 2-heptanone precursor and glycerol as a water activity depressor for the solid-state fermentation of
T. viride
for cheese aroma production. Cultures were grown in Petri dishes at 20° C. Sporulation was visible on the 9th day.
Toyama et al (J. Ferment. Technol.; (1983, 61, 4, 409-11) reported a sporulation medium for
T. reesei
QM 9414, to be used for the production of protoplasts. Kennedy-M-J; Davies-R-J; Surrey-M-R; Reader-S-L; Hoefakker-P-C, Australia's. Biotechnol; (1995) 5, 6, 349-54 reported preparation of biological control agents based on
Serratia entomophila, Bacillus thuringiensis
, Pestalotia species,
Truncatella augustata, Trichoderma viride
, Trichoderma species., cricket-paralysis virus, flock-house virus. The report focuses on the 4 areas of process expertise required to develop and produce a commercial biological control agent: (a) culture medium design, (b) scale-up of the biological production system, (c) downstream processing of the product and (d) extending the shelf life of the biological control agent.
Nigam P. (Process-Biochem.; (1994, 29, 5, 337-42) reported a medium containing diluted molasses solutions of 3-4% sugar concentration for
T. viride
QM 9414
, T. reesei
Rut-C-30 NRRL 11460 under submerged fermentation conditions (SF) performed in flasks in 100 ml medium with agitation at 180 rpm for 5 days.
Culture media for two-stage culture of
T. longibrachiatum, T. viride, T. aureoviride
, AN:
91-13819, CA: Moscow-Tech.Inst.Food-Ind. were reported. The culture medium (pH 4.0-4.2) used in stage I had the following composition (wt. %): wheat bran, 0.3-0.4; additional hydrolysed cotton cake, 0.3-0.4; gibbersib (sic) biomass, 0.45-0.50; NH
4
H
2
PO
4
, 0.25-0.30; K
2
SO
4
, 0.20-0.22; and MgSO
4
, 0.025-0.030. The culture medium used in stage 11 (pH 5.0-5.2) had the following composition (wt. %): beet-root pulp, 2.5-2.8; wheat bran, 0.3-0.4; additional hydrolysed cotton cake, 0.3-0.4; K
2
SO
4
, 0.25-0.30; NH
4
NO
3,
0.25-0.30 and stomach contents hydrolyzate containing reducing compounds, 0.20-0.25.
Abou-Zeid (Bioresource-Technol.; (1991) 37, 3, 239-42) reported the following medium (pH 6) (g/l): cellulosic-source, 10.0; (NH
4
)
2
SO
4
, 2.0; KH
2
PO
4,
1.0; KCl, 0.5; MgSO
4
.7H
2
O, 0.5; MnSO
4
.4H
2
O, 0.05; and FeSO
4
.7H
2
O, 0.005, for the Fermentation of
T. viride
using leaflets and midribs of date palm (
Phoenix dactylifera
) leaves dried at 100-105° C., 144 hr at 30° C. with 200 rpm agitation. Pre-treatment of palm leaflets with 1% NaOH and 5% H
2
SO
4
improved their suitability as C-sources for
T. viride
growth.
The conventional media reported earlier are based on expensive gradients such as yeast extract, malt extract, protein hydrolysates and the like. Such a media are not suited for the economic mass production of these biocontrol agents by fermentation at technical scales.
We have designed medium for the mass production of spores of Trichoderma species, a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and other scantily sporulating species of fungi under solid state, surface and submerged fermentation conditions based on inexpensive agro-based industrial by-products like beet-root pulp, wheat bran, corn-steep liquor and molasses. These agro-waste industrial by products are rich in micro-nutrients and other essential elements like sugars, amino acids providing synergistic effects on the growth and sporulation of these fungi.
OBJECTS OF THE PRESENT INVENTION
The main object of the present invention is to develop a composition for early sporulation of the fungi.
Another main object of the present invention is to develop a composition for profuse sporulation of the fungi.
Yet another object of the present invention is to develop a composition for early and profuse sporulation in Trichoderma species, Aspergillus species, a
Handa Sukhdev Swami
Khajuria Rajinder Kumar
Qazi Ghulam Nabi
Sinha Ram Vilas Parsad
Verma Vijeshwar
Council of Science and Industrial Research
Lilling Herbert J.
Ohlandt Greeley Ruggiero & Perle L.L.P.
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