RAJASTHAN AGRICULTURAL UNIVERSITY, BIKANER...Sri Karan Narendra Agriculture University, Jobner...
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Natural and Chemical Induced Resistance against Sclerotinia Rot of Indian Mustard
[Brassica juncea (L.) Czern & Coss]
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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
Sri Karan Narendra Agriculture University, Jobner
S.K.N. College of Agriculture, Jobner
CERTIFICATE-I
Dated :_______2014
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.
Bansidhar Yadav, grandmother Smt. Mangli Devi, father Sh. Laxmi Narayan, lovely
mother Smt. Mohani Devi, Jiju Bhadhaurji, elder brother Mr. Dinesh, Rajesh and
Ranjeet Yadav, elder sisters Hansa di, younger bro. Mahesh, Rakesh, Ajay, Vinod, Ashok,
Mahendra and Shivam younger sis. Kanchan, Komal, Bhabhi Shakhshi and Lalita and
lovely nephew Aditya and Tanish, whose consistent encouragement and blessing are
beyond my expression that brought me here up to dream without which it could not have
been sketched.
I am also grateful to Sh. Shanker Yadav of Shivam Computer Centre, Jobner, who
typed this manuscript.
Last but not the least, a million of thanks to God. The Almighty who made me to this
task and made every job a success for me.
Place: Jobner
Dated: (ARJUN LAL YADAV)
CONTENTS
CHAPTER NO.
PARTICULARS PAGE NO.
1.
INTRODUCTION
………..
2. REVIEW OF LITERATURE ………..
3. MATERIALS AND METHODS ………..
4. RESULTS ………..
5. DISCUSSION ………..
6. SUMMARY ………..
LITERATURE CITED ………..
ABSTRACT (English) ………..
ABSTRACT (Hindi) ………..
LIST OF TABLES
Table No.
Particulars Page No.
3.1 Different SAR activators and their concentrations ……
3.2 Plant extracts and their concentrations
4.1 Biochemical changes in leaves of healthy and infected
(S. sclerotiorum) plants of Indian mustard varieties
……
4.2 Efficacy of SAR activators against Sclerotinia
Sclerotiorum by poisoned food technique after 7 days
of incubation at 25 + 1 0C
……
4.3 Efficacy of SAR activators against Sclerotinia rot of
Indian mustard
……
4.4 Fungitoxicity of different oil cakes against Sclerotinia
sclerotiorum by poisoned food technique after 7 days
of incubation at 25 + 1 0C
……
4.5 Fungitoxicity of different plant extracts against
Sclerotinia sclerotiorum by poisoned food technique
after 7 days of incubation at 25 + 1 0C
……
LIST OF FIGURES
Figure No.
Particulars Between Page No.
4.1 Biochemical changes (total soluble sugar) in leaves of
healthy and infected (S. sclerotiorum) plants of Indian
mustard varieties
……
4.2 Biochemical changes (total protein content) in leaves
of healthy and infected (S. sclerotiorum) plants of
Indian mustard varieties
……
4.3 Biochemical changes (phenol content) in leaves of
healthy and infected (S. sclerotiorum) plants of Indian
mustard varieties
…….
4.4 Efficacy of SAR activators against Sclerotinia
Sclerotiorum by poisoned food technique after 7 days
of incubation at 25 + 1 0C (in vitro)
…….
4.5 Efficacy of SAR activators against Sclerotinia rot of
Indian mustard (in vivo) …….
4.6 Fungitoxicity of different oil cakes against Sclerotinia
sclerotiorum by poisoned food technique after 7 days
of incubation at 25 + 1 0C (in vitro)
…….
4.7 Fungitoxicity of different plant extracts against
Sclerotinia sclerotiorum by poisoned food technique
after 7 days of incubation at 25 + 1 0C (in vitro)
…….
LIST OF PLATES
Plate
No.
Plate Between
Page No.
1 Pathogenicity test ……
2 Efficacy of different SAR activators against S.
sclerotiorum (in vitro)
…….
3 Fungitoxicity of different oil cakes against S.
sclerotiorum(in vitro)
…….
4 Fungitoxicity of different plant extracts against S.
sclerotiorum (in vitro)
…….
CHAPTER-1 INTRODUCTION
Indian mustard [Brassica juncea (L.) Czern & Coss] is an important oilseed crop. It
belongs to family (Brassicaceae) Crucifereae. Indian mustard is natural amphidiploids
having 36 chromosomes (2n). It is self-pollinated but certain amount of pollination (2-15%)
occurs due to insects and other factors.
The largest cultivation of Brassica crops is done for edible vegetable oil production.
They also play a pivotal role in world’s agricultural economy and are recognized for their
long history of cultivation and varied uses. They are widely cultivated throughout the world
as condiments and spices for improved flavour of human diet and as fodder crop for
livestock feeding. The oilseed Brassica crops are generally grouped as rape and mustard.
Commonly cultivated species are B. compestris var. Sarson Prain. Yellow and brown
sarson; B. campestris var. toria; B. juncea Czern and Coss; rai and Eruca sativa Lam.:
taramira. They are second most important oilseed crops of India after groundnut in terms of
area and production. Indian mustard [Brassica juncea (L.) Czern and Coss] is the most
important in Indian subcontinent because of its relative tolerance to biotic and abiotic
stresses and inherent high yield potential. The black and brown seeds possess about 40
per cent oil content. The oil is mainly used for direct human consumption. However, its use
in different industries can’t be rules out. After the extraction oil, the cake containing over 40
per cent high quality protein is utilized as a feed for livestock and poultry and also as
organic manure for different field and plantation crops.
India is the third largest country in edible oil economy after USA and China. In
respect of rapeseed and mustard production, the country commands a premier position with
an annual production of 7.8 million tonnes from 6.5 million ha area (Anonymous, 2013-14a).
Rapeseed and mustard crops are extensively grown in northern and western parts of India
viz., Rajasthan, Uttar Pradesh, Madhya Pradesh, Haryana, Gujarat, West Bengal, Assam,
Bihar, Punjab and Jammu & Kashmir. Among these, Rajasthan state ranks first both in area
and production i.e., 2.78 million ha and 3.62 million tonnes, respectively with an average
productivity of 1301 kg ha-1
(Anonymous, 2013-14b). In Rajasthan, Tonk district ranks first
both in area and production (2,66,482 lakh ha and 3,85,847 lakh tonnes, respectively)
followed by Sriganganagar, Alwar, Bharatpur and Hanumangarh (Anonymous, 2013-14b).
The problem of diseases and pests is the most important factor causing yield
instability in rape and mustard. Many diseases have so far been reported from different
parts of the world. Among these, diseases caused by fungi take heavy toll of the crop.
Some economic important diseases of rape and mustard in India are leaf spot and blight
(Alternaria brassicae and Alternaria brassicicola), white rust (Albugo candida), downy
mildew (Peronospora brassicae), powdery mildew (Erysiphe cruciferarum), root gall smut
(Urocystis brassicae), stem rot (Sclerotinia sclerotiorum), Rhizoctonia leaf blight
(Rhizoctonia solani), black leaf canker (Leptosphaeria maculans), grey mold (Botrytis
cinerea), Fusarium wilt (F. oxysporum f. sp. conglutinans), Macrophomina wilt (M.
phaseolina), damping off (Pythium butleri), black rot (Xanthomonas campestris pv.
campestris) and mosaic (Saha et al., 1989).
Sclerotinia sclerotiorum (Lib.) de Bary, is a ubiquitous, omnivorous, soil borne and
destructive plant pathogen, inciting disease on more than 400 plant species (Boland and
Hall, 1994). The fungus is an Ascomycete, in the order Helotiales and family
Sclerotiniaceae, characterized by the formation of hard blackish sclerotia, which on
germination produce cup-shaped brown coloured apothecia.
Stem rot or stem blight or white blight or white rot of Indian mustard incited by
Sclerotinia sclerotiorum (Lib.) de Bary is an economically important yield reducing disease
that has been widely reported in the last few years in India and elsewhere. It was earlier
considered to be a disease of minor importance but it has now become a serious disease of
crucifers in some parts of India like Rajasthan, Haryana, Madhya Pradesh and Bihar
(Aggarwal et al., 1997). The disease is monocyclic and symptoms usually appear four to six
weeks after initial infection or at post-flowering stage, when significant damage has already
been done to the crop. Sudden drooping of leaves followed by drying of plants area
characteristics features of the disease. Hyphae from myceliogenically germinated sclerotia
infect basal part of stems causing stem rot. Air-borne ascospores produced by carpogenic
germination of sclerotia during periods of high moisture and favourable temperature
conditions are the major source of inoculums for primary infection in the standing crop. The
petals act as a substrate for ascospore-bearing petals are deposited on leaves. White
cottony, fungal growth is visible near collar portion of the stem with black sclerotial bodies
measuring from 2-20 x 3-7 mm on and inside the stem. When the infected stem of the host
is splitted, large cavities lined by fluffy mycelial growth and numerous black sclerotia of the
fungus are seen which are the main structure responsible for fungus to overwinter and
disseminate.
This disease is gaining importance in the raya-growing areas, which may lead to
disastrous crop failure as the disease incidence was recorded up to 73.8 per cent in some
districts of Punjab and Harayana and at a few locations it went up to 80 per cent (Kang and
Chahal, 2000 and Sharma et al., 2001). Maximum incidence was recorded at Dausa
(29.2%) district in Rajasthan followed by Rohtak (24.8%) districts in Haryana and minimum
7.0 and 7.4 per cent at Jaipur and Bharatpur districts, respectively (Yadav et al., 2013).
Control of this disease mainly depends on fungicides (Tripathi and Tripathi, 2010).
However, fungicidal applications cause hazards to human health and increase
environmental pollution. Induction of host resistance through chemical inducers and use of
resistant cultivars are the effective solutions to overcome the problem. Therefore, keeping
in view all these facts, the investigations were carried out with the following objectives.
(i) To know the chemical basis of susceptibility and resistance in Indian mustard
cultivars against Sclerotinia sclerotiorum.
(ii) To evaluate resistance inducer chemicals against Sclerotinia sclerotiorum in vitro.
(iii) To elicit systemic acquired resistance by chemical inducers against Sclerotinia
sclerotiorum.
(iv) To test the fungitoxicity of organic amendments and phytoextracts against
Sclerotinia sclerotiorum
CHAPTER-2
REVIEW OF LITERATURE
In the beginning, the pathogen was described for the first time from Belgium by
Libert (1837) as Peziza sclerotiorum Libert. Later, it was transferred to a new genus
Sclerotinia by Fuckel (1870) who changed the specific name to Sclerotinia libertiana Fuckel.
Later, de Bary (1884) changed the binomial of Peziza sclerotiorum to Sclerotinia
sclerotiorum. Saccardo (1884) retained the genus Sclerotinia Fuckel. But, Wakefield (1924)
showed it to be inconsistent with the International Rules of Botanical Nomenclature and
cited G.E. Massee as the proper authority for S. sclerotiorum (Lib.) Massee, because he
had used that binomial in 1885, but de Bary used it in his contribution in 1884. Therefore,
the proper name and authority for the fungus seems to be Sclerotinia sclerotiorum (Lib.) de
Bary. Whetzel (1945) described S. sclerotiorum as type spices of Sclerotinia and named 14
other spices, which included S. trifoliorum and S. minor as members of the genus
Sclerotinia. Korf and Dumont (1972), however, suggested the name Whetzelinia in honour
of Professor Whetzel but the special committee of International Association of Plant
Taxonomist recommended that the generic name Sclerotinia be used (Kohn, 1979).
In India, Sclerotinia stem rot reported for the first time by Shaw and Ajerakar (1915)
on several hosts including rapeseed and mustard.
Sclerotinia sclerotiorum reported to be one of the most omnivorous plant pathogen.
It has wide host range that includes 64 families, 225 genera, 361 species and 22 other
cultivars for a total of 383 speceis (Purdy, 1979).
From Rajasthan, the pathogen S. sclerotiorum was first reported on gaillardia (Rai
and Agnihotri, 1970) from Jobner. Later on, it was reported on fennel, potato, sunflower,
pea, brinjal, mustard etc. from other parts of the state (Sehgal and Agrawal, 1971 and Singh
and Agrawal, 1989).
Morrall et al. (1976) reported infection level of Sclerotinia rot up to 62% on Brassica
in Saskatchewan, Canada. Roy and Saikia (1976) reported an outbreak of S. sclerotiorum
from Assam in mustard under cool and wet environmental conditions. Infection at early
growth stages resulted in failure of the crop. Gladders et al. (1991) reported Sclerotinia
epidemics in winter rape oilseed in England and Wales, where the infection level was up to
46%. From trace to 50% losses in seed yield vary with poor quality and quantity of oil has
been reported by Singh (1998) due to Sclerotinia stem rot on mustard.
Management of soil borne pathogen is very difficult but various methods of disease
control viz. chemical, biocides, cultural practices and host plant resistance etc. may reduce
disease incidence.
2.1 To know the chemical basis of susceptibility and resistance in Indian mustard
varieties against Sclerotinia sclerotiorum.
Yarwood (1957) found that bean plants (Phaseolus vulgaris) infected
with (Uromyces phaseoli) showed high accumulation of carbohydrates
around the rust lesions.
Imman (1962) reported that infection of great northern strain 59 bean
(Phaseolus vulgaris) with Uromyces phaseoli typical race 3 resulted in an
increase about two to three folds in soluble sugars and starch, accompanied
by qualitative changes.
Mohamed et al. (1976) found that stems of wheat and barley cultivars
showed the maximum amounts of total carbohydrates in the milk stage then
decreased with the exception of tosson variety in which the maximum
amount was in the tillering stage. Inoculation with stem rust pathogen
accounted to decrease in the amounts of total carbohydrates compared
with uninoculated healthy plants. This decrease was more pronounced in
moderate susceptible or susceptible cultivars than in resistant or moderate
resistant cultivars especially in the milk stage when infection with stem rust
is usually severe.
Basyouni et al . (1976) found that plants susceptible to wheat leaf rust contained
higher level of total phenols, however, the free forms of phenols were higher in amount and
or proportion in the resistant plants. They added that the phenolic level required for
resistance varied from one entry to another and against one race to another. Therefore,
plant phenols and particularly the free forms, were a critical factor in determining adult plant
resistance in wheat to leaf rust.
Helal et al. (1978) found that resistant cucumber variety poinsettia
contained low level of total soluble sugars which prevents further
establishment of Erysiphe cichoracearum within host tissues of the infected
leaves. They also found higher amount of preformed phenols which hinder
fungal infection in the same resistant variety.
Rai et al. (1980) screened cultivars against S. sclerotiorum and the most resistant
and susceptible were selected for study. Healthy stems of the former contained relatively
more phenolics than those of the latter. In infected stems phenolics accumulated at the
infection site in the resistant and there was a relatively low degree of enzyme activity
compared with that in susceptible. The pH was also lowered but to a lesser extent in
resistant than in susceptible infected tissues.
Dhawan (1980) isolated four isolates of S. sclerotiorum and tested on two cultivars
of Chinese mustard which produced significant amount of protease. Protease activity was
greater in the susceptible than in the resistant cultivar.
Kiessling and Hoffmann (1985) reported that inhibition of
photosynthesis, accumulation of starch, glucose and sucrose around the site
of infection until the end of the latent period by infection of rust pathogen on
bean and other crops.
Manners (1989) revealed that sucrose is one of the most important
carbohydrates in plants especially during host parasite interaction as a
transport for carbohydrates satisfying the requirements of leaf and leaf-
borne pathogens.
Tetlow and Farrar (1992) demonstrated a decrease in sucrose
phosphate synthase in Hordeum distichum after infection with Puccinia
hordei. Infection promoted hydrolysis of sucrose to glucose and fructose to
starch.
Scholes et al. (1994) stated and verified the alterations in
carbohydrate status during infection with obligate fungi. Changes in
carbohydrates in the early stages of infection might influence the regulation
of the Calvin cycle.
Wagner and Boyle (1995) investigated the changes in carbohydrate metabolism
and senescence during the development of the bean-rust fungus, Uromyces appendiculatus
(Pers.) Link at different stages of urediospore and teliospore production. Samples were
taken from infected and non-infected bean leaves beginning with the flecking stage. The
unsoluble carbohydrate, sucrose, glucose and fructose content, as well as fluctuations in
soluble acid invertase activity and parameters of leaf senescence were determined. While
fructose remained constant without significant differences between infected leaf sections
and the control. Differences in the other carbohydrates and in soluble invertase activity
could be detected. Parameters indicating leaf senescence were retarded and decreased
with the age, except for peroxidase activity which was enhanced and increased with the age
in infected tissue to a higher degree than in the control .
Lee et al. (2000) compared two Glycine max near-isolines, GL2415 (glyphosate
sensitive) and GL2600RR (glyphosate resistant) for susceptibility to Sclerotinia
sclerotiorum. Results from these studies indicated no differences in fungal growth, disease
development or G. max yield.
Starzycka et al. (2000) reported resistance of 78 top yielding strains of bean against
S. sclerotiorum by inoculating 1551 plants using the “pin-up” method in a field experiment
conducted in Borowo, Poland. They recognized the most resistant (PNG 2170, MA 1615-1,
MZL 236, BK 2466/93, MA 1649-1) and the most susceptible strains (MA 0742-2, PN
610z/98, MZL 189, MZL 201, PN 9011AB/97).
Abd-EL-Magid et al. (2004) revealed that citric acid, tartaric acid, sodium citrate,
sodium salicylate, sodium benzoate and ammonium tartarate decreased the radial growth
of Sclerotium cepivorum Berk, and decreased number of sclerotia. The chemical analysis of
healthy or infected onion bulbs or garlic cloves indicated a higher phenolic content in
healthy onion bulbs than in the infected ones and higher amounts of the amino acids in
healthy samples than infected ones both in onion and garlic.
Ghasolia and Shivpuri (2005) screened 32 rapeseed and mustard genotypes for
resistance to Sclerotinia stem rot (Sclerotinia sclerotiorum) under artificial inoculation
conditions in the field for three consecutive cropping seasons. Among them, eight (Hyola-
401, PBN-9501, PWR-9541, Kiran, RH-9401, RH-492, RW-8410 and PAB-9511) showed
resistant to moderately resistant reaction to S. sclerotiorum.
Guox and Stotz (2007) reported that genotypic differences in susceptibility of
Arabidopsis thaliana to Sclerotinia sclerotiorum have not been reported due to the extreme
susceptibility of this cruciferous plant. To overcome this limitation, two coi1 mutant alleles
conferred hypersusceptibility to S. sclerotiorum. The plant defensin gene PDF1.2 was no
longer induced after challenging the coi1-2 mutant with S. sclerotiorum. Hypersusceptibility
of the coi1-2 mutant to S. sclerotiorum was not correlated with oxalate sensitivity. The
mutants npr1 and ein2 were also hypersusceptible to S. sclerotiorum. Induction of PDF1.2
and the pathogenesis-related gene PR1 was reduced in ein2 and npr1 mutants,
respectively. Actigard, a commercial formulation of the systemic acquired resistance
inducer benzothiadiazole, reduced susceptibility to S. sclerotiorum. Based on histochemical
analysis of oxalate-deficient and wild-type strains of S. sclerotiorum, oxalate caused a
decrease in hydrogen peroxide production but no detectable changes in plant superoxide
production or gene expression.
Jing-Fan et al. (2013) reported that Chinese cabbage (Brassica campestris sp.
chinensis Makino) varieties (003-R-6, Y3-R-4, 109-S-9 and 003-S-7) with different
resistance to Sclerotinia sclerotiorum were selected and used to analyze the physiological
and biochemical changes such as the content of malondialdehyde (MDA), free amino acid
(FAA), the activity of chitinase and beta -1, 3-glucanase. The result showesd that increase
of MDA, FAA in the susceptible variety 003-S-7 was higher than that of resistant variety
003-R-6. On the contrary, the increase of chitinase and beta -1, 3-glucanase activity in the
resistant varieties was higher than that of the susceptible ones.
2.2 To evaluate resistance inducer chemicals against Sclerotinia sclerotiorum in
vitro
Shahda (2000) reported that benzoic acid, salicylic acid and ascorbic acid
significantly reduced the linear growth of the three fungi and spore germination of Fusarium
spp. at 20 mM. The antioxidants used exhibited growth promoting effect where they
increased shoot and root length and dry weight. The three antioxidants significantly reduced
damping off when used as soil drench at 20 mM. The antioxidants were superior to Rizolex-
T either in controlling the disease or in promoting the plant growth.
EL-Ganaieny et al. (2002) evaluated antioxidants like aminobutyric acid (ABA),
potassium salicylate (PS), oxalic acid (OA), salicylic acid (SA) and ascorbic acid (AA) for
their effect on the Fusarium oxysporum, F. solani and F. moniliforme. Mycelial growth was
reduced by the antioxidants. Spore germination was greatly reduced by many tested
chemicals at 8 mM concentration or less. The inhibitory effect of the antioxidants increased
with increasing concentrations. ABA was the only antioxidant which induced protection in all
onion cultivars against all tested Fusarium species.
Hilal et al. (2006) performed a laboratory experiment to evaluate the
antifungal activity of Bion, chitosan, oxalic acid and salicylic acid at 5
concentrations against Sclerotinia sclerotiorum on PDA. All of the
concentrations of the tested compounds significantly reduced linear growth
of S. sclerotiorum compared with the control.
Abdel-Monaim et al. (2012) reported the effectiveness of some chemical inducers
viz. ethephon, hydrogen peroxide (H2O2), mannitol, salicylic acid at three different
concentrations (50, 100 and 200 ppm) on root rot and wilt. All tested chemical inducers
were significantly reduced mycelia linear growth of all tomato root rot and wilt tested fungi
compared with control. The highest decrease in linear growth was noticed with ethephon at
concentration 200 followed by SA at 200 ppm.
Rahamah et al. (2012) evaluated the effect of single and combined application of
calcium ion (Ca2+
), copper ion (Cu2+
) and salicylic acid (SA) on growth and sporulation of
Ganoderma boninense. In poison medium test, T7 (Ca+Cu+SA) showed effective control of
G. boninense in vitro.
2.3 To elicit systemic acquired resistance by chemical inducers against
Sclerotinia sclerotiorum
Shalaby et al. (2001) revealed that the efficacy of helichrysum flower extract, castor
bean oil, aminobutryic acid, potassium salicylate, oxalic acid and salicylic acid in controlling
Macrophomina phaseolina on sesame and sun flower was investigated in laboratory,
greenhouse and field. The evaluated compounds, except helichrysum flower extracts, did
not inhibit the growth of M. phaseolina in vitro. In the greenhouse, all treatments reduced
M.phaseolina incidence and increased plant height, peroxidase activity and IAA content of
sesame and sunflower plants.
Reglinski et al. (2001) stated that salicylic acid (SA) and pre-inoculation with
Sclerotinia sclerotiorum, and the endogenous SA and phenylalanine ammonialayse (PAL)
activity during the period between pathogen inoculation and the onset of systemic
resistance, were investigated under field conditions in Newzealand. Pretreatment of leaves
with SA or pre-inoculation with S. sclerotiorum induced resistance to a challenge inoculation
with S. sclerotiorum 4 days later. Total SA level in the inoculated and Inoculation-1 leaves
did not increase significantly 96 h after inoculation. PAL activity started to increase in the
inoculated leaf after 48 h post-inoculation, and by 96 h was 7 fold greater than the basal
level, while no change was observed in Inoculation- 1 and Inoculation +1 leaves.
Pankaj and Choudhary (2001) tested cupric chloride, ferric chloride, lithium sulfate,
manganese sulfate and sodium molybdate against Sclerotium rot of chickpea. Ferric
chloride and manganese sulfate were the most effective and sodium molybdate was the
least effective. In such effective treatments, the infected host tissue showed marked
reductions in polygalacturanase activity and oxalic acid contents and also moderate
increase in calcium and magnesium levels.
Application of acetyl salicylic acid, amino isobutyric acid, IAA, DL-beta amino-
butyric acid and salicylic acid resulted in lesion-free leaves 72 h after inoculation with
Rhizoctonia solani. Seed and root tip treatment and foliar spray with gamma- amino n-
butyric acid, DL-beta-amino-butyric acid and isonicotinic acid, respectively, gave the highest
control of the disease (Dantre et al., 2003).
Molloy et al. (2004) measured the susceptibility of carrots to Sclerotinia
sclerotiorum after treating with chitosan hydrolysate (0.2%). Sclerotinia infection of carrot
was declined after treating with chitosan hydrolysate due to induced host resistance.
EL-Mougy et al. (2004) revealed that some salts as potassium chloride
,monopotassium sulphate and potassium sulphate are reported to have antifungal activities
against several fungi also used salicylic acid (SA) or acetyl salicylic acid (ASA) as seed
dressing or soil drench which reduced root rot infection of lupine plants under green house
conditions . Furthermore resistance against plant pathogens can be also increased by
salicylic acid or acetyl salicylic acid.
EL-Mougy (2004) evaluated salicylic acid (SA) and acetylsalicylic acid
(ASA) in addition to Rizolex-T as seed dressing or soil drench against lupin
root rot pathogens under greenhouse conditions. No significant reduction in
seed germination was observed when lupin seeds were treated with SA and
ASA up to 2 and 3 g/ kg, respectively. Raising the applied dosages has
reflected negatively on seed germination. SA, ASA and Rizolex-T as seed
dressing or soil drench at the rate of 2, 3 and 3 g/ kg, respectively, have
significantly reduced the percentage of root rot incidence at both pre- and
post –emergence stages of lupin plants growth comparing with untreated
control in artificially infested soil with Fusarium solani, Rhizoctonia solani
and Sclerotium rolfsii. He suggested the use of SA and ASA as seed
dressing or soil drench for controlling such soil borne diseases.
Basha and Chatterjee (2007) reported that foliar application of plant growth-
promoting rhizobacterial strains, i.e. Pseudomonas fluorescens and P. aeruginosa, and two
non-conventional chemicals, i. e. zinc sulfate (10-3
mM) and oxalic acid (4 mM), induced
synthesis of phenylalanine ammonia-lyase (PAL) in wheat. Increased PAL activity followed
by accumulation of different phenolic acids demonstrated that activation of the
phenylpropanoid pathway was one of the reasons for resistance in wheat against
Sclerotinia sclerotiorum and thereby serves as a non-host. Greater accumulation of
phenolics due to increased PAL activity offered protection against diseases (Grey et al.,
1997; Singh et al., 2002, 2003).
EL-Bana (2007) tested antioxidants as seed treatment to induce resistance in chick
pea plants against Sclerotinia sclerotiorum infection. Ability of antioxidants to induce
resistance varied with chickpea cultivars. Malic acid seed treatment was the most
resistance inducer followed by salicylic acid and cumaric acid.
Hathout et al. (2010) applied jasmonic acid as seed soaking that
significantly reduced root rot incidence in bean plants. Production of reactive
oxygen species especially H2O2 was highly increased in both roots and
shoots of the plants infected with Rhizoctonia solani, as compared with non-
infected control. Jasmonic acid significantly increased the total phenolic
compounds in bean roots.
Abdel-Monaim et al. (2012) reported the effectiveness of some chemical inducers
viz. ethephon, hydrogen peroxide (H2O2), mannitol, salicylic acid (SA) at three different
concentrations (50, 100 and 200 ppm) on root rot and wilt diseases incidence as well as
their influence on growth, quantity and quality parameters of tomato plants. On the other
hand, F. solani more affected with chemical inducers than F. oxysporum or R. solani. Under
field conditions, the treatments were accompanied with significant increase in tomato
growth, yield quantity and quality parameters.
2.4 To test the fungitoxicity of organic amendments and phytoextracts against
Sclerotinia sclerotiorum
Meena et al. (2006) proved that application of aqueous garlic clove extract as seed
treatment (1% w/v) followed by a foliar spray at 50 days after sowing could be a user-
friendly, effective and economic non-chemical means of managing Sclerotinia rot of
mustard.
Chattopadhyay et al. (2007) reported that treatment with GR isolate of Trichoderma
harzianum and Allium sativum clove extract caused significant increase in seed germination
and radicle length of Indian mustard by reducing Sclerotinia rot.
Yadav et al. (2009) evaluated ten plant extracts for their antimicrobial
capacity against 5 isolates of S. sclerotiorum. All the plant extracts tested
were found effective in controlling the radial growth of S. sclerotiorum.
However, the Calotropis gigantea and Azadirachta indica extracts were very
effective in reducing the radial growth of S. sclerotiorum.
Tripathi and Tripathi (2009) used different plant extracts viz. Allium
sativum, Eucalyptus globosus, Azadirachta indica, Ocimum sanctum [O.
tenuiflorum], Parthenium hysterophorus, Bougainvillea spectabilis, Lantana
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-
amino butyric acid (100 ppm), Salicylic acid (100 ppm), Hydrogen peroxide (100 ppm), 2,6-
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)
Per cent decrease
Total protein content (mg/g of dry leaf)
Per cent decrease
Phenol content (mg/g of dry leaf)
Per cent increase
Disease incidence
(%)# Healthy* Infected** Healthy* Infected** Healthy* Infected**
Bio-902 11.90 9.50 20.17 20.5 20.00 2.44 4.85 4.92 1.44 30.00 (33.21)
Kranti 12.90 10.13 21.47 22.6 22.10 2.21 5.10 5.34 4.71 10.00 (18.43)
Varuna 10.80 7.20 33.33 21.1 20.00 5.21 3.92 3.98 1.53 36.66 (37.26)
Aravali 11.25 8.75 22.22 22.4 21.70 3.13 4.46 4.52 1.35 21.11 (27.35)
RRN-505 10.90 8.05 26.15 19.9 19.30 3.02 4.62 4.74 2.60 22.22 (28.12)
Navgold 12.40 9.45 23.79 21.9 21.15 3.42 4.45 4.50 1.12 20.00 (26.57)
Manihari 13.02 8.10 37.79 21.6 20.40 5.56 3.95 4.00 1.27 33.33 (35.26)
NRCDR-2 11.25 8.15 27.56 21.7 21.00 3.23 4.58 4.65 1.53 27.77 (31.80)
Laxmi 11.62 9.15 21.26 22.4 21.90 2.23 5.03 5.24 4.17 13.33 (21.41)
RGN-48 13.02 9.20 29.34 19.7 19.10 3.05 4.20 4.32 2.86 24.44 (29.63)
NRCDR-601 11.45 7.90 31.00 21.9 20.80 5.02 4.02 4.10 1.99 30.00 (33.21)
SEm+ 0.350 0.221 - 0.518 0.523 - 0.156 0.105 - 0.426 CD (p=0.05) 1.033 0.653 - 1.529 1.543 - 0.460 0.311 - 1.257
* Leaves collected 30 DAS and inoculum applied after it ** Leaves collected 45 DAS # Average of three replications
Table:4.2 Efficacy of SAR activators against Sclerotinia sclerotiorum by poisoned food technique after 7 days of incubation at 25 + 1 0C
SAR activators Per cent growth inhibition at various concentration (ppm)
100 200 500 Mean
β-amino butyric acid (BABA)
77.77
(61.87)
86.66
(68.58)
92.00
(73.57)
88.14
(68.01)
Salicylic acid 90.37
(71.92)
100.00
(90.00)
100.00
(90.00)
96.79
(83.93)
Hydrogen peroxide 16.66
(24.09)
38.88
(38.57)
100.00
(90.00)
51.85
(50.89)
2,6-Di Chloroisonicotinic
acid
24.44
(29.63)
38.52
(38.36)
80.00
(63.43)
35.43
(43.81)
Azoxystrobin 18.48
(25.46)
50.22
(45.13)
73.33
(58.91)
47.34
(43.17)
Carbendazim (Standard
check)
100.00
(90.00)
100.00
(90.00)
100.00
(90.00)
100.00
(90.00)
Control 0.00
0.00
0.00
0.00
SEm+ CD (p=0.05)
A 1.00 2.87
C 0.66 1.88
AxC 1.74 4.97
* Average of three replications
Figures given in parenthesis are angular transformed value
Table:4.3 In vivo efficacy of SAR activators against Sclerotinia rot of Indian mustard SAR activators Concentration used
(ppm) Seed soaking Foliar spray Seed soaking-cum- foliar spray
Disease intensity
(%)*
Per cent disease control
Disease intensity
(%)*
Per cent disease control
Disease intensity (%)*
Per cent disease control
β-amino butyric acid 100 37.10 (37.52)
28.93 30.00 (33.21)
45.45 23.90 (29.27)
50.33
Salicylic acid 100 35.32 (36.46)
32.34 27.60 (31.69)
49.82 21.90 (27.90)
54.49
Hydrogen peroxide 100 39.60 (39.00)
24.14 33.70 (35.49)
38.73 27.00 (31.31)
43.89
2,6-Di Chloroisonicotinic acid
100 40.60 (39.58)
22.22 36.00 (36.87)
34.55 27.90 (31.88)
42.02
Azoxystrobin 2000 26.00 (30.66)
50.19 21.60 (27.69)
60.73 14.65 (22.50)
69.56
Carbendazim (Standard check)
1000 10.82 (19.20)
79.27 12.65 (20.83)
77.00 6.32 (14.56)
86.87
Control - 52.20 (46.26)
0.00 55.00 (47.87)
0.00 48.12 (43.92)
0.00
SEm+ 0.69 - 0.87 - 0.41 - CD (p=0.05) 2.19 - 2.77 - 1.31 -
* Average of four replications
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.
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