Natural and Chemical Induced Resistance against Sclerotinia Rot of Indian Mustard [Brassica juncea (L.) Czern & Coss] ljlksa [czsfldk tfUl;k ¼fy-½ tuZ ,oa dkWl] ds LDysjksfVfu;k lM+ u ds fo:} uSlfxZd ,oa jlk;u mRizsfjr izfrjks/kdrk Thesis Submitted to the Sri Karan Narandra Agriculture University, Jobner In partial fulfillment of the requirement for the degree of Master of Science In the Faculty of Agriculture (Plant Pathology) By Arjun Lal Yadav 2014
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Natural and Chemical Induced Resistance against Sclerotinia Rot of Indian Mustard
[Brassica juncea (L.) Czern & Coss]
ljlksa [czsfldk tfUl;k ¼fy-½ tuZ ,oa dkWl] ds LDysjksfVfu;k
This is to certify that Mr. Arjun Lal Yadav has successfully
completed the comprehensive examination held on ----------2014 as required
under the regulation for Master’s degree.
(K.S. SHEKHAWAT) Professor & HEAD
Department of Plant Pathology S.K.N. College of Agriculture,
Jobner
Sri Karan Narendra Agriculture University, Jobner
S.K.N. College of Agriculture, Jobner
CERTIFICATE-II
Dated :________2014
This is to certify that the thesis entitled “Natural and Chemical
Induced Resistance against Sclerotinia Rot of Indian Mustard
[Brassica juncea (L.) Czern & Coss]” submitted for the degree of Master
of Science in the subject of Plant Pathology embodies bonafide research
work carried out by Mr. Arjun Lal Yadav under my guidance and
supervision and that no part of this thesis has been submitted for any other
degree. The assistance and help received during the course of investigation
have been fully acknowledged. The draft of the thesis was also approved by
the advisory committee on 09.12.2014.
(K.S. Shekhawat)
Professor & HEAD (R.P. Ghasolia) Department of Plant Pathology Major Advisor S.K.N. College of Agriculture, Jobner
(G.L. KESHWA) DEAN
S.K.N. College of Agriculture, Jobner
Sri Karan Narendra Agriculture University, Jobner
S.K.N. College of Agriculture, Jobner
CERTIFICATE-III
Dated :______2014
This is to certify that the thesis entitled “Natural and Chemical
Induced Resistance against Sclerotinia Rot of Indian Mustard
[Brassica juncea (L.) Czern & Coss]” submitted by Mr Arjun Lal Yadav to
Sri Karan Narendra Agriculture University, Jobner, in partial fulfillment of the
requirements for the degree of Master of Science in the subject of Plant
Pathology after recommendation by the external examiner, was defended
by the candidate before the following members of the examination
committee. The performance of the candidate in the oral examination on his
thesis has been found satisfactory. We therefore, recommend that the thesis
be approved.
(R.P. Ghasolia) (A.C. Mathur) Major Advisor Advisor
(K.C. Kumawat) (K. Ram Krishna) Advisor Director Education Nominee (K.S. Shekhawat) Professor & HEAD (G.L. KESHWA) Department of Plant Pathology Dean S.K.N. College of Agriculture, S.K.N. College of Agriculture, Jobner Jobner Approved
DIRECTOR EDUCATION S.K.N. Agriculture University, Jobner
Sri Karan Narendra Agriculture University, Jobner
S.K.N. College of Agriculture, Jobner
CERTIFICATE-IV
Dated : _____ 2014
This is to certify that Mr Arjun Lal Yadav of the Department of Plant
Pathology, S.K.N., College of Agriculture, Jobner has made all corrections/
modifications in the thesis entitled “Natural and Chemical Induced
Resistance against Sclerotinia Rot of Indian Mustard [Brassica juncea
(L.) Czern & Coss]” which were suggested by the external examiner and
the advisory committee in the oral examination held on ______2014. The
final copies of the thesis duly bound and corrected were submitted on
______2014 and forwarded herewith for approval.
(R.P. Ghasolia) Major Advisor
(K.S. Shekhawat) PROFESSOR & HEAD
Department of Plant Pathology S.K.N. College of Agriculture, Jobner
(G.L. KESHWA)
DEAN S.K.N. College of Agriculture, Jobner
APPROVED
DIRECTOR EDUCATION SKNAU, Jobner
ACKNOWLEDGEMENT
In the ecstasy. I express my esteem and profound sense of gratitude to Dr. R.P.
Ghasolia, major advisor, Asstt. Professor, Department of Plant Pathology, S.K.N. College
of Agriculture, Jobner, for his valuable and inspiring guidance, constant encouragement
and keen interest during the course of present investigation and preparation of this
research report.
I convey the feeling of gratitude to the members of my advisory committee Dr. A.C.
Mathur, Professor, Deptt. of Plant Pathology, Dr. K.C. Kumawat, Professor, Deptt. of
Entomology and Dr. K. Ram Krishna, Professor, Deptt. of Plant Breeding & Genetics,
Dean P.G. Nominee for the help rendered during the course of investigation.
I also deep and heartful greatfulness to Dean, S. K. N. College of Agriculture, Jobner
and Dr. K.S. Shekhawat, Head , Deptt. of Plant Pathology for providing necessary
facilities during the course of investigation.
I am again ecstatic to express my inherent sense of gratitude to Dr. R.R. Ahir, Dr. S.
Godika, Dr. Mahaveer Choudhary and all the staff members, Deptt. of Plant Pathology,
S.K.N. College of Agriculture, Jobner whose cooperation made this investigation smooth
and easy.
Down the memory lane. I am very much thankful to my dear friends Sajjan, Dinesh,
Ganpat, Ramchandra, Pawan my seniors Rekha ji, Sanju ji, Mukesh ji, Suresh ji, Gopal ji
and my lovely juniors Jitendra, Vishambar, Sukh Lal, Roshan, Rajesh, Raj kumar,
Pramod and Suresh with whom I went hand in hand during my study and research work.
My vocabulary falls short to express heartiest regards to my grandfather Sh.
camara, Datura stramonium, Calotropis procera and Capsicum annum
against Sclerotinia sclerotiorum causing stem rot of Indian mustard
(Brassica juncea). Maximum inhibition of pathogen colony growth was
observed in Allium sativum (71.11%) followed by Azadirachta
indica (50.37%), O. sanctum (38.15%) and C. procera (22.96%). The
remaining plant extracts were ineffective in controlling the mycelial growth of
the pathogens.
Yadav (2009) reported that five botanical plant extracts (1%) viz., Allium sativum, A.
cepa, Azardirachta indica, Calotropis procera and Eucalyptus globosus were tested for their
biopesticidal activity on mustard diseases. A. sativum (garlic) and Eucalyptus were found
more effective than Calotropis (aak) and Azardirachta indica (neem).
Tripathi et al. (2011) tested six organic amendments viz., sunflower cake, safflower
cake, groundnut cake, mustard cake, neem cake and farmyard manure against Sclerotinia
sclerotiorum and found maximum inhibition of mycelial growth (30.37%) in sunflower cake
while minimum (2.22%) in groundnut cake.
CHAPTER-3 MATERIALS AND METHODS
3.1 To know the chemical basis of susceptibility and resistance in Indian mustard varieties against Sclerotinia sclerotiorum.
Sclerotinia rot infected samples of Indian mustard were collected from
Bassi Tehsil of Jaipur. Stem rot pathogen was isolated from infected tissues.
The pathogen was identified as Sclerotinia sclerotiorum on the basis of
morphological, cultural characters and pathogenicity was proved (Plate 1).
3.1.1 Bio-chemical studies
The following experiments were conducted in order to study the effect
of Sclerotinia rot on some biochemical constituents (proteins, carbohydrates,
phenols) of mustard varieties, namely, Bio-902, Kranti, Varuna, Manihari,
Aravali, RRN-505, Navgold, NRCDR-2, Laxmi, RGN-48 and NRCDR-601.
These varieties were grown in pots (9x12 inches) with three replication in
cage house. Healthy leaves from above varieties were collected 30 days
after sowing (DAS) than washed and dried in shade for biochemical
analysis. After it, inoculum (multiplied on sorghum grains) were added @ 20
g/pot. Leaves from infected plants were collected 45 DAS and disease
incidence were also noted.
For the quantitative estimation of primary metabolites following different
protocols were used.
3.1.1.1 Determination of total soluble sugar
Leaf material (0.1-0.5 g) without midrib were extracted in 10 ml of
80% ethanol in a mortar pestle, the extract obtained was centrifuged at 8000
rpm for 10 min. Extraction was repeated four times with 5 ml of 80% ethanol
each time and supernatants were collected into same beaker. Volume of the
extract was made to 50 ml with 80% ethanol. A 0.1 to 0.5 ml of aliquot from
supernatant was added to 4.0 ml of Anthrone reagent (0.2% : 200 mg
anthrone dissolved in 100 ml H2SO4, prepared fresh before use) and the test
tubes were placed in ice cold water. The intensity of colour was read at 620
nm on spectrophotometer. A standard curve was prepared using glucose
(100 µg/ml) (Dubois et al., 1951).
3.1.1.2 Determination of phenol content
One ml of supernatant was taken from ethanol extract prepared for
total soluble sugar analysis (3.1.1.1) and evaporated to dryness in water
bath. One ml of milipore water in each test tube and 0.5 ml of Folin &
Ciocalteu reagent (1:1 with water) was added and kept for three min. After
this, 2 ml of 20% Na2CO3 was added and mixed thoroughly. The tubes were
placed in boiling water for exactly one minute and cooled in ice water. The
absorbance was read at 650 nm against a reagent blank (Malik and Singh,
1980).
A standard graph was prepared using pyrocatachol ranging between 0-25 µg concentration.
The amount of phenols present in the sample was calculated as: Phenol (mg/g) = Sample O.D. × Standard O.D. × Dilution factor Where O.D. = Optical density
3.1.1.3 Determination of total protein
Protein concentration of extract was estimated by method of Lowry et al. (1951).
(a.) Reagents for Lowry’s method
(i) Solution A: 2% Na2CO3 in 0.1 N NaOH
(ii) Solution B: (a) 1% CuSO4.5H2O solution
(b) 2% sodium potassium tartrate solution
Working solution of B: Prepared fresh before use by mixing equal
volume of solution B (a) and B (b).
(iii) Solution C: Prepared fresh before use by mixing 50 ml of solution A
and 1ml of working solution of B.
(iv) Solution D: Folin & Ciocalteu reagent (1N).
(b.) Procedure
Extracts of different samples (25µl) were taken in separate test tubes
and volume was made up to 1 ml in each tube with milipore water. A tube
with 1 ml of water served as blank. Five ml of solution C was mixed in each
tube by vortexing and kept for 10 min. Then 0.5 ml of solution D (Folin &
Ciocalteu reagent) was added in each tube and vortexed. The tubes were
allowed to stand at room temperature for 30 min. Absorbance was read at
660 nm. A standard curve was prepared using bovine serum albumin (BSA)
in the concentration range of 10-80 µg.
3.2 To evaluate resistance inducer chemicals against Sclerotinia
sclerotiorum (in vitro)
The following systemic acquired resistance (SAR) activators in addition
to carbendazim (standard check) were used.
Table :3.1 Different systemic acquired resistance activators
S.No. SAR activators
1. β-amino butyric acid
2. Salicylic acid
3. Hydrogen peroxide
4. 2,6-Di Chloroisonicotinc acid
5. Azoxystrobin
The SAR activators (Table 3.1) in addition to carbendazim (standard
check) were evaluated with three (100, 200 and 500 ppm) concentrations
against the pathogen under laboratory conditions to find out their relative
efficacy in inhibiting the growth of the pathogen in culture by poisoned food
technique (Schmitz, 1930). Requisite quantity of each chemical was
incorporated in sterilized two per cent potato dextrose agar medium,
thoroughly mixed by shaking prior to pouring in sterilized Petriplates and
were allowed to solidify. These Petriplates were inoculated with 5 mm disc
of four day old culture in the centre of the plate and incubated at 25 + 10C.
Each treatment was replicated three with a suitable control. The efficacy of
chemicals in each treatment and average of three replications was
calculated. Per cent inhibition over control was calculated by the following
formula (Bliss, 1934).
C - T
Per cent inhibition over control = ———— x 100
C
C = growth of fungus in control
T = growth of fungus in treatment
3.3 To elicit systemic acquired resistance by chemical inducers against
Sclerotinia sclerotiorum (in vivo)
The experiment was carried out in earthen pots (9 x 12 inches) with
cultivar T-59 (Varuna). The pathogen multiplied on sorghum grains at 25 + 1
0C for one week was used as the soil inoculum. Prior to sowing, pots were
sterilized with copper sulphate solution and filled with sterilized soil (soil :
vermicompost 3 :1). The soil was sterilized at 1.045 kg/cm2 for one hour for
three consecultive days. Varuna, the susceptible variety of Indian mustard
was sown in these pots with four replications. The SAR activators viz., β-
Di Chloroisonicotinc acid (100 ppm), Azoxystrobin (2000 ppm) and carbendazim (1000
ppm) were tested by applying as seed soaking (for 30 minutes), foliar spray (30 DAS) and
seed soaking-cum-foliar spray.
These pots were inoculated with inoculum multiplied on sorghum
grains 45 days after sowing. For inoculation the upper 5 cm layer of soil of
each pot was thoroughly mixed with inoculum @ 20 g/pot. The pots were
covered with polythene bags and kept for 24 hours in cage house.
To assess the stem rot intensity the following slightly modified
disease rating (0-4) scale (Lesovoi et al., 1987 and Sansford, 1995) was
followed.
Grade/numerical
scale
Description/lesion lenghth on stem
0 Healthy (no visible lesion)
1 0.1-2.0 cm lesion length on stem
2 2.1-4.0 cm lesion length on stem
3 4.1-6.0 cm lesion length on stem
4 > 6 cm lesion length on stem or complete dried plant
The length of lesion on infected stem was considered for recording the disease intensity (Sharma, 1987). The infected area was calculated from 10 randomly selected plants at 80 DAS. In each pot and the average for each treatment was worked out. The intensity was calculated using the formula of Wheeler (1969).
Sum of individual ratings Per cent disease intensity = ----------------------------------------------------------- x 100 No. of plants observed x maximum disease rating
3.4 To test the fungitoxicity of organic amendments and phytoextracts against Sclerotinia sclerotiorum
3.4.1 Organic amendments
Five organic amendments viz., neem cake, mustard cake, castor
cake, groundnut cake and sesame cake were screened in vitro to evaluate
their inhibitory effect on radial growth of the pathogen. Hundred g
oil cake was taken in 1000 ml water and preserved in earthen pot for
extraction. Pots were wrapped by polythene bags to preserve moisture. Oil
cake extract was filtered with cheese cloth, mixed @ 3% in PDA in conical
flask and autoclaved. Twenty ml PDA was poured in each sterilized
Petridish and allowed for solidify. The each plate was inoculated with 5 mm
diameter bit of 7 days old culture of fungus. Inoculated plates were
incubated at 25 + 10C for 7 days. The linear growth of test fungus was
recorded and per cent growth inhibition was calculated as per Bliss (1934)
formula referred under 3.2.
3.4.2 Phytoextract
To find out the fungitoxicity of six plant extracts (Table 3.2) against
the pathogen were evaluated. Hundred gram from each was collected and
washed 2-3 times with water and allowed to dry at room temperature
(25±10C) for six hours. Before extraction leaves of each plant (100g) were
crushed separately with 100 ml sterilized distilled water. The extract was
filtered through muslin cloth and centrifuged at 5000 rpm for 30 min. The
extract were then sterilized by passing them through a Millipore filter using a
swimming filter adapter.
The extract of each plant species was diluted in order to achieve
three concentrations viz., 5, 10 and 15 per cent. Petri plates containing PDA
supplemented with different phyto-extracts, each with three concentrations
and replicated three times were inoculated with 5-day-old culture (5 mm dia
disc). A suitable check (without plant extract) was also maintained. Fungal
colony was measured after 7 days of incubation at 25 + 1 0C. The linear
growth of test fungus was recorded and per cent growth inhibition was
calculated by Bliss (1934) formula referred under 3.2.
Table : 3.2 Plant extracts and their concentrations
S.No. Common Name
Botanical Name Part used
Concentration (%)
1 Garlic Allium sativum Clove 5, 10, 15
2 Neem Azadirachta indica Leaves 5, 10, 15
3 Tulsi Ocimum sanctum Leaves 5, 10, 15
4 Alstonia (devil’s tree)
Alstonia scholaris Leaves 5, 10, 15
5 Ginger Zingiber officinalis Rhizome 5, 10, 15
6 Turmeric Curcuma longa Rhizome 5, 10, 15
CHAPTER-4
RESULTS
4.1 To know the chemical basis of susceptibility and resistance in
Indian mustard cultivars against Sclerotinia sclerotiorum
Biochemical studies: Changes in total soluble sugars, total protein content
and total phenols were estimated in leaves of healthy and infected (S.
sclerotiorum) plants of different Indian mustard varieties (Table 4.1).
The resistance of a plant as a result of host pathogen interaction
involves morphological and biochemical changes which depends upon the
plant response to infection. The fungal infection induces the oxidative and
hydrolytic reactions along with hormonal imbalance in the host tissue
affecting the normal metabolism.
4.1.1 Total soluble sugar
Total soluble sugar (Table 4.1 and Fig. 4.1) in leaves of healthy and
infected (Sclerotinia sclerotiorum) plants of Indian mustard varieteis was
estiamted at 30 & 45 DAS, respectively. The total soluble sugar was
decreased (20.17 – 37.79%) in all the infected plants as compared to
healthy ones. The total soluble sugar was decreased maximum (37.79%) in
infected plants of Manihari variety followed by Varuna (33.33%), NRCDR-
601 (31.00%), RGN-48 (29.34%) and minimum in Bio-902 (20.17%) as
compared to healthy ones. Minimum disease incidence (Table 4.1) was
recorded in Kranti (10%), as it also resulted in minimum reduction in total
soluble sugars.
4.1.2 Total protein content
Total protein content (Table 4.1 and Fig. 4.2) in leaves of healthy and
infected (Sclerotinia sclerotiorum) plants of Indian mustard varieteis was
estiamted at 30 and 45 DAS, respectively. The total protein content was
decreased in all infected plants as compared to healthy ones. The total
protein content was decreased maximum (5.56%) in infected plants of
Manihari variety followed by Varuna (5.21%), NRCDR-601 (5.02%) and it
was found minimum in Kranti (2.21%).
4.1.3 Total phenol
Phenol content (Table 4.1 and Fig. 4.3) in leaves of healthy and
infected (Sclerotinia sclerotiorum) plants of Indian mustard varieteis was
estimated at 30 and 45 DAS, respectivley. The total phenol content was
increased in infected plants as compared to healthy ones. Phenol content
was increased maximum (4.71%) in infected plants of Kranti variety followed
by Laxmi (4.17%) and it was increased minimum in Novgold (1.12%),
Manihari (1.27%), Aravali (1.35%), Bio-902 (1.44%) and Varuna (1.53%).
It is cleared (Table 4.1) that variety Kranti showed lowest disease
incidence (10.00%) as it had highest amount of total phenol (5.10 mg/g dry
leaf) in healthy and infected leaves (5.34%). Varieties like Novgold,
Manihari, Aravali, Bio-902, Varuna had low level of increased phenol content
and showed maximum disease incidence (20.00 to 36.66%).
4.2 To evaluate resistance inducer chemicals against Sclerotinia
sclerotiorum in vitro
The efficacy of SAR activators in addition to carbendazim was
evaluated against Sclerotinia sclerotiorum on PDA by poisoned food
technique. The data suggested (Table 4.2, Fig. 4.4 and Plate 4.2) that
increase in concentration of the SAR activators caused increased inhibition
of mycelial growth of the fungus. Among these, salicylic acid was found cent
per cent inhibitory at 200 ppm. This was followed by β-amino butyric acid
(77.77, 86.66 and 92.00 %) at 100, 200 and 500 ppm, respectively.
Azoxystrobin (18.48, 50.22 and 73.33%) and 2, 6-Dichloroisonicotinic acid
(24.44, 38.52 and 80.00%) were found to be least effective at 100, 200 and
500 ppm, respectively against Sclerotinia sclerotiorum.
4.3 To elicit systemic acquired resistance by chemical inducers
against Sclerotinia sclerotiorum (in vivo)
4.3.1 Seed soaking
A perusal of data (Table 4.3 and Fig.4.5) revealed minimum disease
intensity with azoxystrobin (26.00%) followed by salicylic acid (35.32%), as
compared to control (52.20%). Maximum reduction in disease intensity over
control was osberved with azoxystrobin (50.19%) followed by salicylic acid
(32.34%) over control. β-amino butyric acid (28.93%) was found at par with
salicylic acid. Minimum reduction in disease intensity was observed in 2, 6-
Dichloroisonicotnic acid (22.22%).
4.3.2 Foliar spray
Perusal of data (Table 4.3 and Fig. 4.5) revealed a similar trend of
results as in 4.3.1. The highest reduction in disease intensity over control
was observed in azoxystrobin (60.73%) followed by salicylic acid (49.82%),
β-amino butyric acid (45.45%), hydrogen peroxide (38.73%) and minimum in
2, 6 Dichloroiso-nicotinic acid (34.55%).
4.3.3 Seed-cum-foliar spray
A perusal of data (Table 4.3 and Fig. 4.5) revealed minimum disease
intensity in azoxystrobin (14.65%) followed by salicylic acid (21.90%) and β-
amino butyric acid (23.90%) over control (48.12%).
Maximum reduction in disease intensity over control was observed in
azoxystrobin (69.56%) followed by salicylic acid (54.49%), β-amino butyric
acid (50.33%) and minimum in 2, 6 Dichloroiso nicotinic acid (42.02%).
4.4 To test the fungitoxicity of organic amendments and
phytoextracts against Sclerotinia sclerotiorum
4.4.1 Organic amendements
Efficacy of five organic oil cakes was tested in vitro against
Sclerotinia sclerotirum. The castor cake (Table 4.4) was found significantly
superior over all the tested oil cakes with maximum (44.44%) inhibition of
mycelial growth of Sclerotinia sclerotiorum over control followed by neem
cake (40.00%). Groundnut cake was found least effective (5.55%) in
inhibiting mycelial growth of S. sclerotiorum (Table 4.4, Fig. 4.6 and Plate
4.3).
4.4.2 Phytoextracts
The efficacy of six plant leaf extracts (Table 4.5) was tested in vitro at
three concentrations viz., 5, 10 and 15 per cent against S. sclerotiorum on
PDA by poisoned food technique. Among six plant extracts, extract of garlic
cloves was found most effective in inhibiting mycelial growth (52.22, 65.66
and 88.00 %) of S. sclerotiorum at 5, 10 and 15 per cent, respectivley
followed by neem (50.74, 67.20 and 80.0%) over control. Extract of Alstonia,
tulsi and turmeric were found least effective in inhibiting mycelial growth of
S. sclerotiorum over control.
All the cocentrations (5, 10 and 15%) of garlic extract were found
significantly superior over each other while 10 and 15 per cent of Alstonia,
tulsi and turmeric were found at par to each other.
Table:4.1 Biochemical changes in leaves of healthy and infected (S. sclerotiorum) plants of Indian mustard varieties
Varieties Total soluble sugar (mg/100mg of dry leaf)
Figures given in parenthesis are angular transformed value
Table:4.5 Fungitoxicity of different plant extracts against Sclerotinia
sclerotiorum by poisoned food technique after 7 days of incubation at
25 + 1 0C
Plant extract Part used Per cent growth inhibition at different
concentration
(%)*
5 10 15 Mean
Garlic Clove 52.22
(46.27)
65.66
(54.13)
88.00
(69.73)
68.63
Turmeric Rhizome 25.96
(30.36)
61.11
(51.42)
67.82
(55.42)
51.63
Neem Leaves 50.74
(55.39)
67.20
(45.17)
80.00
(63.43)
66.01
Ginger Rhizome 41.00
(39.82)
56.20
(48.56)
71.04
(57.44)
56.08
Tulsi Leaves 45.07
(42.17)
49.30
(44.60)
60.26
(50.92)
51.54
Alstonia Leaves 35.82
(36.76)
50.93
(45.53)
62.96
(52.51)
49.90
Control - 0.00 0.00 0.00
0.00
SEm+ CD (p=0.05)
E 1.44 4.11
Con. 0.94 2.69
E x Con. 2.49 7.12
* Average of three replications
Figures given in parenthesis are angular transformed value
Table:4.4 Fungitoxicity of different oil cakes against Sclerotinia sclerotiorum by poisoned food technique after 7 days of incubation at 25 + 1
0C
Organic amendments Concentration (%)
Radial mycelial
growth (mm)*
Per cent growth
inhibition
Mustard cake 3.00 83.00 7.77
(16.17)
Neem cake 3.00 54.00 40.00
(39.23)
Castor cake 3.00 50.00 44.44
(41.81)
Groundnut cake 3.00 85.00 5.55
(13.63)
Sesame cake 3.00 70.00 22.22
(28.12)
Control - 90.00 0.00
(0.00)
SEm+ - - 0.28
CD (p=0.05) - - 0.88
* Average of three replications
Figures given in parenthesis are angular transformed value
CHAPTER-5
DISCUSSION
Indian mustard [Brassica juncea (L.) Czern & Coss] has gained importance in
different parts of Rajasthan as a potential oilseed crop and is grown in almost every district
of the state. This crop suffers from vagaries of fungal, bacterial and viral diseases. Among
the fungal diseases, Sclerotinia rot caused by Sclerotinia sclerotiorum (Lib.) de Bary, earlier
considered to be a minor disease, is now becoming increasingly destructive and widely
damaging in recent years. Particularly in areas of heavy soils receiving four or more
irrigation (Ghasolia et al., 2004).
The total soluble sugars level decreased in all the cultivars of Indian mustard after
infection by S. sclerotiorum. After infection by S. sclerotiorum, varieties showing higher
reduction in total soluble sugars, expect few, were highly susceptible & depicted higher
disease incidence while varieties with less reduction were moderately resistant. These
results are in accordance with the findings of Teltow and Farrar (1992) and Jaypal and
Mahadevan (1968). Teltow and Farrar (1992) reported post infectional decrease in sugar
levels in Puccinia infected barley may be due to rapid hydrolysis of sugars during
pathogenesis through enzymes secreted by the pathogen. The invading pathogens may
utilize the sugar leading to decrease in its content. Mohamed et al. (1976) also recorded
reduction in total amount of carbohydrates in infected (Puccinia sp.) wheat and barley plants
compared with healthy plants. This reduction was more pronounced in moderate susceptible
cultivars than in resistant or moderate resistant cultivars. After infection by S. sclerotiorum,
Jing-fan et al. (2013) estimated higher biochemical changes in the susceptible varieties of
Chinese cabbage than that of resistant ones.
Total protein content in infected leaves was lower as compared to healthy ones.
After infection, varieties showing higher reduction in total protein content, except few were
highly susceptible and showed higher disease incidence while varieties with less reduction
were moderately resistant. These results are confirmatory with the findings of Sempio and
Marte (1968), who reported that protein content was decreased in susceptible variety of
beans against rust pathogen. During host pathogen interaction, between barley and
Septoria sp. amino acid act as a substrate for the pathogen (Titarenko et al., 1993).
Plants have developed an arsenal of defense mechanisms to protect themselves
against pathogen attacks. In the present investigation, phenol content in leaves of infected
plants was higher as compared to healthy ones. It is cleared that varieties showing high
increase in phenol content after infection were highly resistant and showed lowest disease
incidence while varieties with low increase were highly susceptible. Singh et al. (2011)
concluded that presence of phenolics in high concentration in the plant cell gives them
resistance to pathogens. After infection by pathogen plant cells synthesize phenol oxidizing
enzymes that oxidize phenols to toxic quinines. Hence the activity of these enzymes
increases in infected cell. Basyouni et al. (1976) also reported increase in the phenolic
content of wheat cultivars after infection. Helal et al. (1978) reported increase in the amount
of total phenols in both resistant and susceptible cultivars of cucumber infected with
Erysiphe cichoracearum. Various workers (Patil and Dimond, 1967; Ravise and Trique,
1972 and Rai et al., 1980) recorded increase in the amount of phenolic substances in
different host pathogen system after infection. Our results are also in accordance with the
findings of Basha and Chatterjee (2007), who reported that increased level of phenolics
activate the phenylpropanoid pathway which is one of the reasons for resistance in wheat
against S. sclerotiorum. They also explained that phenylpropanoid pathway may be utilized
to induce resistance in the hosts of S. sclerotiorum by biotic and abiotic agents (Singh et al.
2011).
All the SAR activators (β-amino butyric acid, salicylic acid, hydrogen peroxide, 2, 6
Dichloroiso nicotinic acid and azoxystrobin) in addition to carbendazim were tested at 100,
200 and 500 ppm concentration and inhibited mycelial growth of S. sclerotiorum. Our
observations are in agreement with El. Ganaieny et al. (2002), Shahda (2002), Hilal et al.
(2006), Abdel-Monaim et al. (2012) and Rahaman et al. (2012).They reported that all the
tested abiotic agents were found significantly superior in reducing linear growth of many
fungal pathogens including S. sclerotiorum.
Protection of plants against pathogens depends on constitutive and induced
defense mechanisms (Nurnberger and Lipka, 2005). Extensive research has shown that
biotic and abiotic agents including salicylic acid play a key role in local and systemic
acquired resistance (SAR) to biotrophic and necrotrophic pathogens (Durrant and Dong,
2004; Gaffney et al., 1993; Thomma et al., 1998 and Conarth et al., 2002). Amongst
different SAR activators used in the present investigation were applied through seed
soaking, foliar and seed-cum-foliar spray. In these methods, seed soaking-cum-foliar spray
of SAR activators was found most effective to control disease by reducing disease intensity,
followed by foliar spray and seed soaking alone. All SAR activators tested were able to
reduce the disease intensity significantly over control. Azoxystrobin, a fungicide-cum-SAR
inducer was the most effective in reducing the disease intensity followed by salicylic acid, β-
amino butyric acid, hydrogen peroxide and 2, 6 Dichloroisonicotinc acid. These results are
in agreement with the results of Dantre et al. (2003), Abdel-Monaim et al. (2012), El Bana
(2007), El-Mougy et al. (2004) and Shalaby et al. (2001). They reported effectiveness of
various SAR inducer in disease control against many fungal pathogens including S.
sclerotiorum.
Extracts of five oil cakes were screened in vitro for fungitoxicity against S.
sclerotiorum and observed that castor and neem cake were found most effective in inhibiting
mycelial growth. Earlier workers have also been reported oil cakes as a source for inhibition
of the fungal growth. Chand and Rai (2008), reported saw dust, castor cake and neem cake
significantly superior in inhibiting mycelial growth of S. sclerotiorum. Inhibitory effect of six
organic amendments on mycelial growth of S. sclerotiorum have also been reported by
Tripathi et al. (2011).
Six plant extracts namely garlic, neem, ginger, turmeric, tulsi and Alstonia were
tested at 5, 10 and 15 per cent concentration and inhibited mycelial growth of S.
sclerotiorum in vitro. Garlic gave maximum inhibition of mycelial growth at higher
concentration. Similar results have been observed by Tripathi and Tripathi (2009) while
working with S. sclerotiorum in vitro. Extracts of ten plants have also been evaluated in vitro
against S. sclerotiorum by Yadav et al. (2009) and reported effective in inhibiting mycelial
growth
CHAPTER-6
SUMMARY
Indian mustard (Brassica juncea L.) has gained importance in different parts of
Rajasthan as potential oilseed crop and is grown in almost every district of Rajasthan. This
crop suffers from many disease among which stem rot caused by Sclerotinia sclerotiorum
(Lib.) de Bary, has become a serious problem in recent years in Rajasthan and in other
mustard growing parts of India.
Total soluble sugars, total protein content and phenol content have received
considerable attention in relation to resistance in plants against diseases. The biochemical
estimation is carried out to study the host parasite relationship in Indian mustard cultivars
infected with S. sclerotiorum. Leaves of infected and healthy plants were taken in to
consideration. Low disease incidence was recorded in varieties showing decreased level of
sugars and protein content and increased level of phenols after infection. This may impart
resistant power to plants to fight against pathogen.
Studies on the relative efficacy of SAR activators were tested in vitro and found that
salicylic acid was observed to be most effective in inhibiting mycelial growth followed by β-
amino butyric acid.
To activate defense mechanism of plants, five SAR activators were evaluated as
seed soaking, foliar and seed soaking-cum-foliar sprays. Azoxystrobin proved to be most
effective against Sclerotinia sclerotiorum followed by salicylic acid, β-amino butyric acid,
hydrogen peroxide and 2, 6 Dichloroiso nicotinic acid in reducing per cent disease intensity.
Castor cake was found most effective followed by neem cake in inhibiting mycelial
growth in vitro.
Garlic extract was found most effective followed by neem extract in inhibiting
mycelial growth in vitro.
Natural and Chemical Induced Resistance against Sclerotinia Rot
of Indian Mustard [Brassica juncea (L.) Czern & Coss]
Arjun Lal Yadav * Dr. R. P. Ghasolia ** (Research Scholar) (Major Advisor)
ABSTRACT
Sclerotinia rot infected samples of Indian mustard were collected from Bassi tehsil of Jaipur district. Pathogen isolated, purified and identified as Sclerotinia sclerotiorum & its pathogenicity was proved.
Biochemical changes in total soluble sugars, total proteins content and phenol content played very important role in relation to resistance in plants against disease. An increase in total phenols and decline in total soluble sugars and total protein content were observed in the leaves of infected plants with S. sclerotiorum compared to the healthy ones which impart resistance to plants.
Five SAR activators were tested by poisoned food technique and inhibited the growth of fungus. Salycilic acid checked mycelia growth completely at 200 ppm followed by β-amino butyric acid
SAR activators were used as seed soaking, foliar spray and seed soaking-cum-foliar spray. Azoxystrobin and salicylic acid were observed to be most effective in reducing the disease intensity.
Among oil cakes, castor cake was found most effective followed by neem cake in inhibiting mycelial growth.
Garlic extract was found most effective in inhibiting mycelial growth followed by neem.
* Post graduate student, M.Sc. (Ag.), Department of Plant Pathology, S.K.N. College of Agriculture, Jobner.
** Thesis submitted in partial fulfillment of the requirement for M.Sc. (Ag.), degree in Plant Pathology under supervision of Dr. R.P. Ghasolia, Assistant Professor, Department of Plant Pathology, (SKN Agriculture University, Jobner), S.K.N. College of Agriculture, Jobner, Jaipur.