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(11) Patent Number: KE 303
P    (45) Date of grant: 23/02/2009
Kenya Industrial Property Institute.
(12) PATENT
(51) Int.CI.8: A 01114/00
 
(21)Application Number:
(22) Filing Date: (31)Priority Number:
(73)    Owner(s):
(72) Inventor(s)
(74)    Agent/address for correspondence:
(54) Title:
(57) Abstract:
 
KE/P/ 1999/ 000284
26/06/1999
(32) Date: (33) Country:  SECRETARY DEPARTMENT OF BIOTECNOLOGY of , C. G .0. COMPLEX,LODI ROAD, NEW DELHI 110003, INDIA, India and DIRECTOR G.B PANT INSTITUTE OF HIMALAYAN ENVIRONMENT & DEVEOPMENT of , KASI-KATARMAL, ALMORA U . P. INDIA, India
ANITA PANDEY and DR.LOK MAN SING PALNI
Ndungu Njoroge & Kwach Advocates, P.O. Box 41546-00100, Nairobi
 
ABSTRACT
This    inVenxion rwagwww to a method    of
hardening of tissue culture raised tea plants using antagonistic bacteria. The process comprises in 5    . the    steps    of    preparing    a    suspension of
 antagonistic microorganism (bacteria) having inhibition properties to isolated pathogenic fungi. The bacterial suspension so obtained is introduced into sterile soil. The tissue culture raised plants are
10        placed into said sterile soil having said organism.
The microorganism used are Bacillus subtilis or Pseudommnes corrugate.
 
I LD OF INV NT ION
This    invention relates to a method    of
hardening of tissue culture raised tea plants using soil microbes.
5    BACKGROUND OF INVENTION
Tissue culture raised tea plants experience a high degree of mortality during or following laboratory to land transfer. The tissue culture raised plants often experience low survival and poor
10    establishment, mainly due to extreme differences
between the in vitro and ex vitro environment conditions. The procedure adopted for acclimatization of tissue culture raised plants have not been very satisfactory in providing quality transplants for the
15    greenhouse    and subsequently for the field. Apart
from the various abiotic ( fa biotic) causes, one major probable cause of high degree of mortality of such asceptically raised plants is sudden exposure of these plants (particularly the root system) to
: 2 :
 
microbial communities    (including minor and major
pathogens)    present    in the soil.    Plants,    raised
using    tissue culture    techinques, are maintained
under aseptic conditions and probably do not possess
5 sufficient resistance to defend against the soil micorbial communities. As a result, a large number of such plants, during transplanting, are attacked by soil microorganisms (especially fungi) and are killed.
10    OBJECTS OF THE INVENTION
An object of the invention is to propose a method of hardening of tissue culture raised tea plants through the use of antagonistic bacteria.
Still another object of this invention is to 15    propose a method for highly improved survival or
tissue culture raised tea plants following    transfer
to soil.
: 3 :
 
Further    and advantages    of this
invention will be more apparent from the ensuing description.
DESCRIPTION OF THE INVENTION
5    According to this invention there is provided
a method of hardening of tissue culture raised
tea plants using antagonistic bacteria comprising
in the steps of preparing    a suspension of
antagonistic    microorganism    (bacteria)    having
10    inhibition properties to    isolated pathogenic fungi
introducing of bacterial suspension into sterile soil,
placing    the tissue culture raised plants into said
sterile    soil    having    said    organism,    said
microorganism being Bacillus subtilis or Pseudomones 13    -corrugate.
Briefly, the    invention    resides    in    first
identifying    and    isolating    the    antagonistic
microorganism    having the    inhibition properties
against pathogenic (harmful) fungi.    The step of
20    identification and isolation is described subsequently
herein. However, briefly the step of    identification
: 4 :
 
and isolation comprises in preparing a fungal isolate
suspension of    the various isolated fungi, said
suspension being prepared in sterile water.    The
suspension is spread on an agar plate and    spot
5    incoculated with bacteria at various locations and
incubated for a period of one week. During the period of incubation, both the fungal organisms and microbes (bacteria) grow. If there is a growth of antagonistic microorganism, the fungal growth would
10    be    negligible    (inhibited)    and    whereby    the
antagonistic    bacteria    having    the    inhibition
properties    is identified and isolated.
A    suspension    is    prepared    of    the
identified antagonistic microbe and introduced    into
15    sterile soil. The tissue culture raised plants is
transferred into such a soil.
Various    steps    of the process    are now
described:
 
Isolation and Purification of Bacteria and fungi: Isolation of bacteria and fungi were carried out from tea soils of established and young tea rhizosphere, collected from various
5 tea growing locations. About 200 bacterial and 100 fugnal isolates were obtained using Tryptone Yeast Extract and Potato Dextrose agar/Mycological agar, respectively.
Following    repeated subculture, single colonies
10    were obtained    and the    purified    isolates were
0
stored at 4 C on agar slants.    Bacteria were
isolated    on the    basis    of    their    colony
characteristics. Similarly, the test fungi were also isolated from the    tea rhizosphere.
15    Petridish assay for screening of antagonistic
bacteria:    For    testing    antifungal    activity,    a
fungal    lawn of the test fungus was made    on
Carrot    potato    agar plates.    Eight    bacterial
isolates,    were    then    inoculated    using    spot
20    inoculation    method on these plates.    Following
: 6 :
 
O
incubation at 28 C for 2-4 days, the plates were observed for inhibition zones. Bacteria causing inhibitio zones above 20 mm were considered to be strongly antagonistic and four of them were selected
5    for bioassay.
The    DionPy:    The tea    plants    (Chinery
type, Camellia sinensis 1. (0. kuntze), raised through
somatic embryogenesis on a full strength modified
Murashige and Skoog (1962) medium were used for the
10    bioassay.    Four promising    antagonistic    bacterial
isolates,    selected    on the    basis    of    above
mentioned    petridish    assay, were    used    for
inoculation of tissue culture raised    tea plants.
Suspension culture of bacterial    isolate(s)    was
15    raised in Tryptone Yeast extract broth.    Autoclaved
(i.e., sterile) soil (pH 5.6) was filled in    plastic
pots (8 cm ht. x 7 cm dia.; each containing    approx.
175 g soil). Soil was inoculated by adding    3 ml.
suspension (of one of the    four selected bacteria)
4    5
20    containing    10 to 10 cells per ml. Tissue culture
raised plants    were transplanted (one per pot) in
: 7
 
control    (uninoculated)    as well as    in    treated
(inoculated) pots.    Five treatments with following
details, were taken under considerations
(1)    (control): plants transplanted    in Autoclaved
5    soil    without bacterial inoculation; (2,3,4 and 5):
plants    transplanted in autoclaved soil    inoculated
with    bacterial isolates 1, 2, 3 and 4    respectively
(all    singly).    Tea    plants, in sets a    ten, were
selected    for each treatment.    Observations on
10    survival    and microbial activities    in the control
and inoculated soil were recorded.
Petridish    assay for    antifungal    activity
against fungi isolated in the bioassay experiment:
Plants showing diseased symptoms or poor vigour
15    were harvested and isolations were carried out from
the rhizosphere and rhizoplane on mycological    agar.
The    isolated    fungi were identified as Fusarium
oxysporum,    Trichoderma hamatum and Syncephalastrum
racemoseem.    These fungi were tested against the
20    four    bacterial    inoculants by spot    inoculation
 
method.    Two of the    bacterial isolates exhibited
strong    antifungal activity and resulted    in near
100% survival of tea plants. One of these    bacteria
(isolate    4) also    exhibited    considerable    growth
5    promotion properties (Table 1.)
TABLE 1
Table    1.    Effect of bacterial    inoculation    on
survival and plant growth of tissue culture raised tea plants one year    following transfer to soil.

10    Treatment        %survival    shoot height(mm)        leaf no.
Control            50    16.71    + 1.89    4.00    + 0.35
Bacterial    isolates    1    100    18.43    + 2.36    3.29    + 0.48
Bacterial    isolates 2    70    16.86    + 1.73    3.00    + 0.40
Bacterial    isolates    3    80    15.86    + 2.39    3.86    + 0.71
15Bacterial    isolates    4    100    31.41    + 6.33    5.43    + 0.67

 
9
 
Microbial    analyses of control and    inoculated
rhizoptane and rhizosphere in the bioassay experiment:
Soil    samples were collected    and    analysed for
bacterial    and fungal population    using    serial
5 dilution technique. Nutrient agar and mycOlogical agar were used for such enumerations, respectively. Plating of the infected roots of control plants
on mycological    agar plates,    exhibited    heavy
colonization    by    Fusarium oxysporum,    and    the
10        rhizosphere soil of these plants was colonized by
three fungi, namely F. oxysporum, Trichoderma hamatum
and Syncephalastrum racemoseem, resulting in colony
5
forming    units upto 10 cells/ml.    The bacterial
population    in    this case was relatively smaller.
15 Contrary to this, in selected bacteria inoculated treatments, no fungal colony was observed on soil dilution plates and the rhizoplano and rhizophere were largely colonized by bacterial population.
 
10
 
TABLE 2
Table    2.    Influerftia pf bactitial inoculation on
development of rhizosphere microbial communities (cfu/ml)    around tissue culture raised tea plants
5    one    year following    their transfer to soil.
Treatment    Bacterial counts    Fungal counts
    3    5
Control    22 x 10    9 x 10
4
Bacterial isolates 1    62 x 10
4
Bacterial isolates 2    26 x 10
4
109acterial isolates 3    28 x 10
4
Bacterial isolates 4    48 x 10
- = not deleted
    Identification of promising bacterial:    The
two    bacteria (isolate nos. 1 and 4) resulting    in
15    100X    survival were    texonomically    identified    as
: 11    :
 
Bacillus    Subtilis (associate of    established    tea
rhizosphere) and Pseudomonas corrugate (associate of
young tea rhizosphere). The identification was based
on    cultural,    morphological,    biochemical    and
5    physiological properties.
Reference is made    to Fig. 1 of    the
accompanying drawings which illustrates a petridish
assay having a fungal lawn with the    test bacteria.
The drawing reflects the growth of bacteria A and
10    without any    fungal    growth in the    vicinity or
proximity of said bacteria A.
I t
 
CLAIM
1.    A method of hardening of    tissue culture raised
tea plants using antagonistic bacteria comprising
in the steps of preparing    a suspension of
5    antagonistic    microorganism    (bacteria)    having
inhibition properties to    isolated pathogenic fungi
introducing of bacterial suspension into sterile    soil,
placing    the tissue culture raised plants into said
sterile    soil    having    said    organism,    said
10        microorganism being Bacillus subtilis or Pseudomones
corrugate.
13
 
 
 
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