MSc.

Agriculture is heavily dependent on the use of chemical fertilizers. However,
excessive and unbalanced use of these inputs may lead to environmentalpollution
and health hazards. Biofertilizers are biological fertilizers which play a key role in
maintaining productivityand sustainability of soil, thereby protecting the
environment. Hence, a study was undertaken to isolate, screen and evaluate free
living diazotrophic bacteria to develop an efficient biofertilizer.
Rhizosphere soils and plant samples were collected from ten healthy black
pepper gardens of different locations of Wayanad district. A total of 43 free living
diazotrophic bacteria were isolated on three different N-free media (Jensen’s agar,
Beijerinckia agar and Ashby’s agar). Maximum population of nitrogen fixers was
obtained on Jensen’s agar. Twenty predominant isolates were selected, including
eleven isolates from rhizosphere soil and nine isolates from phylloplane, based on
their growth characteristics on N-free media for further evaluation.
Twenty selected isolates were screened under in vitro condition for growth,
nitrogen fixation and acid tolerance. As pH was lowered from 7.0 to 5.0, a reduction
in growth and nitrogen fixation was observed. However, among the isolates, two
from the rhizosphere (NKdS and NPS-1) and two from phylloplane (NKPV-2 and
NPPV) performed better.
Twenty selected isolates were screened in vitro for plant growth promoting
activities like production of IAA, HCN, siderophore and ammonia.The antagonistic
activity of all the twenty isolates was tested against three soil borne plant pathogens
Fusarium oxysporum, Rhizoctonia solaniand Ralstonia solanacearum. Isolates NkdS,
NPPV and NKS-1 were found to exhibit antagonistic activityagainst Rhizoctonia
solani.Six isolatesinhibited the growth ofRalstonia solanacearum. None of the
isolatesrecorded antagonistic activity against Fusarium oxysporum.
Four most promising diazotrophs including, two rhizosphere isolates (NKdS
and NPS-1) and two phylloplane isolates (NPPV and NKPV-2) were selectedfor in
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planta evaluation, based on nitrogen fixation ability, tolerance to acidic pHand PGPR
activities. 16S rDNA sequence analysis was carried out and promising isolates were
identified asMicrobacterium sp. (NKdS and NPPV); Cellulosimicrobium sp. (NPS-1)
and Brevundimonas sp. (NKPV-2).
In planta evaluation was carried out with tomato as the test crop. These
isolates were applied alone, in combination with 75% N and with 75% N + 50%
FYM. Diazotrophs were inoculated as seed treatment, seedling dip and soil/foliar
application. Seeds treated withKAU Azotobacter formulation triggered faster
germination compared to all other treatments. However, after 7 days of sowing,
maximum number of seedling emergence (90.24%) was recorded in seeds treated
with Cellulosimicrobium sp.
Plant height, number of leaves, fresh weight and volume of root, fresh weight
and dry weight of plants,yield per plant, average fruit weight and number of fruits
were observed to be maximum in treatment T12 (Microbacterium sp.+ 75% N + 50%
FYM). T11 (Cellulosimicrobium sp. +75% N + 50% FYM) recorded maximum girth
of stem, leaf area and minimum days to flowering.
Total nitrogen content of index leaf and plant was maximum in T12
(Microbacterium sp. + 75% N + 50% FYM). The same treatment recorded maximum
nitrogen content in soil at 60DAS. At harvest, T11 (Cellulosimicrobium sp. + 75% N
+ 50% FYM) recorded highest nitrogen content in soil.
Population build up of bioinoculants in soil was higher in presence of
FYM.Endophytic colonization was higher in root, in case of soil application of
bioinoculants and higher in leaves, in case of foliar spray.
The study revealed that novel, free living diazotrophic bacteria like
Microbacterium and Cellulosimicrobium could be exploited as biofertilizer
formeeting 25% of the nitrogen requirement. Results also indicated the importance of
application of organic manure along with the isolates, for their best performance.