MSc

The research work entitled “Characterization of novel strains of soil-inhabiting
Trichoderma spp. and their utilization in soil-borne disease management” was conducted
during the period 2019-2021 at the Department of Plant Pathology, College of Agriculture,
Vellayani, Thiruvananthapuram, Kerala. The study aimed at characterization of novel strains
of Trichoderma spp. obtained from virgin forest soils of Kerala; and their evaluation against
major soil-borne diseases of tomato and vegetable cowpea.
Two reference strains viz., T. harzianum (NBAIR strain) and T. asperellum (KAU
strain) along with 11 different isolates of Trichoderma spp. from the previous study were used
to carry out the present study. All the isolates showed white coloured mycelial growth except
for isolate TRML-1 which showed creamish white coloured mycelium. The textures of the
colonies were sparse, spreading and cottony or fluffy growth at centre. Moreover, the
sporulation and growth pattern also varied among the isolates which showed spores with
varying shades of green. The spore characters including the size and shape of the spores of all
the isolates were observed under the microscope at 100x magnification. Spores of isolate
TRML-1 (4.448 µm x 4.172 µm) were the largest while the spores of isolate TRSN-1 (2.197
µm x 2.881 µm) were the smallest. The shape of the spores varied from globose, sub-globose,
ovoidal or ellipsoidal.
Pathogenicity tests to prove Koch’s postulate revealed that symptoms of Fusarium
wilt (yellowing, drying of foliage followed by wilting of the plant) occurred at five days of
inoculation of the pathogen. Pre-emergence damping off symptoms were seen as water soaked
lesions leading to rotting of the radicle thus preventing the germination of the seeds while
post-emergence damping off exhibited softening of the tissues of collar region leading to
toppling of the seedlings at about 13 days of pathogen inoculation. In the pathogenicity test,
77.78 per cent disease incidence was observed in Fusarium wilt of vegetable cowpea while
70.73 per cent and 100 per cent incidence was observed for pre- and post-emergence damping
off of tomato.
The radial growths of all the Trichoderma isolates as well as of both the pathogens
were recorded. The isolates TRMW-2, TRPN-3, TRPN-11, TRPN-14 and TRPN-15 were fast
growing, covering the Petri plate at three days after inoculation (DAI). Among the pathogens,
it was observed that P. aphanidermatum completed its growth at 3 DAI while F. oxysporum
was slow growing fungus that completed its growth at 9 DAI. In vitro screening of
Trichoderma isolates against both pathogens was carried out. Antagonistic characters viz.,
antibiosis, lysis and overgrowth along with per cent inhibition and antagonistic index were
calculated based on which five potential isolates were selected to carry out further studies.
Isolates TRMW-2, TRPN-3, TRPN-11, TRPN-15 and TRPN-17 showed all the three
antagonistic properties and high antagonistic index against F. oxysporum; the highest
inhibition (47.78%) being shown by the isolate TRPN-3. Similarly isolates TRMW-2, TRKR2, TRPN-15 and TRPN-18 showed all the three antagonistic properties and high antagonistic
index against P. aphanidermatum; the highest inhibition being exhibited by the isolates
TRPN-7, TRPN-18 and TRML-1 (70%). Volatile metabolites produced by the isolates TRPN14 and TRPN-17 showed the highest inhibition on F. oxysporum (71.25%) and
P. aphanidermatum (27.78%) respectively.
RAPD-PCR of genomic DNA of seven isolates of Trichoderma spp. was done using
four primers viz., OPA-02, OPA-03, OPA-09 and OPA-10. The dendrogram showed two
major groups; the first with TRPN-3, TRPN-17, TRKR-2, T. asperellum (KAU strain) and
T. harzianum (NBAIR isolate), and the second with isolates TRPN-11 and TRMW-2.
Molecular identification of ten isolates of Trichoderma spp. was also done using ITS-PCR.
The best matching sequences from GenBank were recorded and phylogenetic tree was
constructed. The isolates were identified as T. harzianum (TRMW-2, TRPN-3, TRPN-14,
TRPN-17), T. koningiopsis (TRKR-2), T. lixii (TRPN-11, TRPN-15) and T. asperellum
(TRPN-18).
Talc based formulations of all the isolates applied as seed treatment and as an
additive in potting medium brought about successful control of pre-emergence and postemergence damping off, among which TRKR-2, TRMW-2 and TRPN-11 were more effective
compared to the treatments with other isolates for both pre- and post-emergence damping off.
The population of Trichoderma spp. in potting medium in different treatments of the in vivo
experiment ranged from 8 x 104
to 12 x 104
cfu g-1
.