STUDIES ON THE VARIABILITY OF Rhizoctonia solani Kühn. INCITANT OF RICE SHEATH BLIGHT DISEASE AND ITS MANAGEMENT
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Date
2024-05-16
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ACHARYA N G RANGA AGRICULTURAL UNIVERSITY
Abstract
The present investigation was carried out to study the variability of
Rhizoctonia solani Kühn . incitant of rice sheath blight disease and its
management. Survey for occurrence and distribution of rice sheath blight disease
was conducted in West Godavari, East Godavari, SPSR Nellore, YSR Kadapa
and Chittoor districts of Andhra Pradesh during Kharif 2019. Among all the
villages surveyed, the highest per cent disease incidence was observed at
Penumanchili village (45.66 %) of West Godavari district and the lowest disease
incidence was recorded at Koppolu (11.31%) in YSR Kadapa district.
A total of thirty pathogen isolates were isolated from the disease samples
collected during survey and their pathogenicity was proved in pot culture studies
by following typha bit inoculation method. Sheaths from healthy plants in
disease- infected field were collected during the survey and thirty bacterial
endophytes were isolated from the samples.
The cultural variability among the 30 isolates of test pathogen, R. solani
was studied on PDA medium. Based on the growth after 72h incubation, ten
isolates were categorized into fast growers (65-69 mm growth) and twenty
moderate growers (60-64mm growth). Based on the pigmentation of the colony
the cultures were assigned to five groups. i.e, ten isolates (Group I- white), three
isolates (Group II-Yellowish white), seven isolates (Group III-pale brown), six
isolates (Group IV – brown) and four isolates (Group V - dark brown). There was
a significant variation among the isolates in the time taken for initiation of
sclerotia, which ranged from 5- 9 days, hyphal width varied from the lowest 13.59
μm to the highest 31.66 μm. The colour of sclerotia produced by different isolates
was white initially and became dark brown with maturity. Number of sclerotia
produced ranged from the lowest 3 to the highest 89. Based on the sclerotia
location in the fungal colony, the R. solani isolates were divided into three groups.
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The first group with sclerotia formed in the aerial surface, the second group with
sclerotia embedded in the fungal mycelium. The third group with both aerially
formed and embedded sclerotia. Pattern of the sclerotia formation was also varied
among isolates. Size of the sclerotia ranged from 0.8 to 2.8 mm. Based on texture
of sclerotia, isolates were categorized into two groups i.e, smooth and rough.
Three rice varieties with varying degree of tolerance to sheath blight
disease, viz. NLR-34449 (susceptible), IR-64 (moderately susceptible), Tetep
(moderately resistant) was selected for pathogenic variability studies of R. solani
isolates. Relative lesion height (RLH) per centage was recorded 10 days after
inoculation with pathogen. On NLR- 34449 variety, isolates WAK, NNA, NPC
showed highly virulent reaction. WAP, NPP, YVP, YVV, YPL, YPD, YPS and
CSS were moderately virulent, however WAA, WTJ, WTM, WTT, ERR, ERV,
ERG, EAA, EAM, EAD, NNK, NNN, NPK, YVK, CSA, CSM, CYY, CYK and
CYP isolates showed less virulent reaction. On IR- 64 variety, isolate WAK,
NNA showed highly virulent reaction. NPC showed moderately virulent response
and rest of all the isolates showed less virulent reaction. On Tetep variety, WAK
and NNA showed moderately virulent reaction and rest of the isolates showed less
virulent reaction. Considering the reaction on all the three rice varieties,
isolate WAK showed the highest RLH i.e., 54.66%, 49.03%, and 39.46% on
NLR 34449, IR64 and Tetep respectively. WAK isolate was used for further in
vitro and field management studies.
RAPD analysis was conducted to know the molecular variability among
R. solani isolates. Among 20 primers used, eight primers namely; OPA-07, OPA 10, OPB-17, OPC-11, OPC-19, OPE-06, OPF-14 and OPG-11, gave reproducible
polymorphism among the 30 isolates. The observed polymorphism among the
isolates was 100 per cent in all eight primers. The PIC (Polymorphic Information
Content) values were ranged from 0.36 (OPC-11) to 0.49 (OPG-11) with an
average PIC value of 0.44 wherein all the primers studied had showed very high
variation among the isolates. Genetic relatedness among the R. solani isolates was
showed through UPGMA cluster analysis dendrogram. The grouping pattern of
the isolates in different clusters indicated that there was less variability among the
isolates of the same mandal of one district as they were grouped under same sub cluster except few isolates YVK, YVV, CSM, CYY, YPL, EAD, YVP, NPC and
ERR which have separated from their respective mandal isolates and grouped
with different sub clusters indicating more variability thus they might had
undergone more evolution than others. Hypergeometric probability was calculated
among the ten isolates of test pathogen R. solani. Highly virulent to moderately
virulent isolates were selected for this studies. WAK, NNA, NPC are highly
virulent WAP, NPP, YVP, YVV, YPL, YPD, YPS, CSS are moderately virulent.
When the isolates were studied through hypergeometric probability pattern of
accidental matching bands gave least probability value (0.014286) between
NPC×YVP isolates indicating their accidental similarity is less and they are very
close each other and represented in single cluster in dendrogram. Similarly, the
next least probability value 0.0833333 was observed between YPS and YPD
isolates which are isolated from YSR Kadapa district indicating their close
relationship. The highest probability value was between WAK and CSS isolates
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i.e., 0.978571 indicating that their accidental similarity is more. This means that
they have very little association or they are totally different isolates.
Eight different fungicides were tested at different concentrations against
R. solani were significantly superior over control in checking the mycelial
growth. At tested concentrations, 100 % inhibition was observed in all the
fungicide treatments except pencycuron. Pencycuron showed decreased
inhibition with decreased concentration, 94.77% inhibition at 1500 ppm followed
by 93.84 % at 1000 ppm and 92.11% at 500 ppm.
Bacterial endophytes were tested against R. solani by dual culture test.
NBE-4 bacterial isolate showed highest inhibition of 54.28%. Hence it was
selected for characterization studies and in vivo studies like compatibility. NBE4
was rod shaped when observed under compound microscope (1000X) and gram
positive in Gram staining test. 16Sr DNA region of potential endophyte NBE4
was amplified using primers 27F and 1492R. 16SrDNA gene sequence of the
potential endophyte was compared with gene sequences available at NCBI
database through BLASTn search. The gene sequence of the strain NBE4
endophytic bacteria exhibited highest identity of 99.46% with Bacillus sp. strain
MR-1/2 (MG548383.1) and its species name was not identified yet. The
sequence of the endophyte was submitted to NCBI gene bank and got accession
number OK655750. The molecular identity of the strain was confirmed as genus
Bacillus species, hence given name as Bacillus sp. NBE4 and it formed a cluster
with B. toyonensis, B. mobilis, B. pacificus and B.cereus when a phylogenetic
tree was constructed with type strains of genus Bacillus using MEGA (ver. 7)
software, which depicts that its sequence was more similar with these species.
Eight different fungicides evaluated using poisoned food technique were
used to test their compatibility with NBE4 endophyte by using Agar well
Diffusion method. Among the tested fungicides, pencycuron 250 SC,
azoxystrobin 18.2% + difenoconazole 11.4% SC, iprobenfos 48% EC, showed
0% inhibition of endophytic bacteria i.e., on par at all the concentrations with
control (0% inhibition). Among these three, azoxystrobin18.2% +difenoconazole
11.4% SC was selected for field trials because pencycuron showed less per cent
inhibition against the R. solani when compared to azoxystrobin18.2%
+difenoconazole 11.4% SC. In addition, azoxystrobin18.2% + difenoconazole
11.4% SC not only effective against sheath blight but also against other major
rice diseases when compared to iprobenfos 48% EC.
Efficacy of certain resistance inducing chemicals salicylic acid, benzoic
acid, naphthalene acetic acid, jasmonic acid @ 50, 100, 150 ppm against R.
solani were tested in pot culture. Plants treated with salicylic acid showed less
disease severity of 20.80% and 20.76% at 100 and 150 ppm concentration,
respectively, and both are statistically at par, whereas at 50 ppm concentration
disease severity was 22.80% which was significantly higher than the other two
concentrations. Induction of defense-related enzymes like PAL (Phenylalanine
Ammonia Lyase), Peroxidases and Polyphenol oxidases due to application of
resistance -inducing chemicals were estimated at 2,4,6 and 8 days after
inoculation. There was significant change in production of these enzymes before
application to after application indicating that these chemicals have ability to
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increase defense enzyme activity in rice plants and activity was increased up-to 6
days and there after decreased gradually. The highest and significant activity of
enzymes was found in salicylic acid (SA) treatment and the disease severity also
recorded less in this treatment.
To know the efficacy of chemicals and bioagent under field conditions an
experiment was conducted in both Kharif 2020 and Rabi 2020-21 on NLR-34449
(Susceptible variety) in Randomized Block Design (RBD).
In the Kharif, Per cent disease incidence in all the treatments were
significantly less as compared to control 63.28% at 70 DAT. Among all the
treatments, the lowest per cent disease incidence was recorded in treatment T9
(seed treatment with carbendazim 1g l-1 + seed treatment with bacterial
antagonist + root dipping with bacterial antagonist before transplanting + foliar
spray with azoxystrobin 18.2% + difenoconazole 11.4% SC @ 1000 ppm + foliar
spray of salicylic acid @ 100 ppm) 26.45% and T7 (seed treatment with
carbendazim 1g l-1 + seed treatment with bacterial antagonist + root dipping with
bacterial antagonist before transplanting + foliar spray with azoxystrobin 18.2% +
difenoconazole 11.4% SC@ 1000 ppm) 26.50% which are at par each other and
their disease reduction over control was 58.20% and 58.12% respectively.
In the Rabi season, per cent disease incidence in all the treatments were
significantly less as compared to control 65.99% at 70 DAT. Among all the
treatments, the lowest per cent disease incidence 27.73%, 28.00% was recorded
in treatment T9 and treatment T7 respectively and both were at par, their disease
reduction over control was 57.97 and 57.56% respectively