efficiency of trichoderma viride against leaf spot of cabbage.pptx

Pre submission
on
Efficacy of Trichoderma viride and selected botanical oils against Alternarial leaf spot (Alternaria brassicae)
disease of cabbage
Advisor:
Dr. (Mrs.) Shashi Tiwari
Assistant Professor
Department of Plant Pathology
Submitted by:
CH.V S Jagadish Kumar
PID. No.- 19MSPP064
M.Sc.Agriculture
(PlantPathology)
Sam Higginbottom University of Agriculture, TechnologyAndSciences
Prayagraj, Uttar Pradesh, 211007.

SAC Name and Designation Contact Information
Chairman Prof. (Dr.) Gautham Ghosh
Dean of Naini Agriculture Institute
SHUATS
Gautham.ghosh@shiats.edu.in
Advisor Dr. (Mrs.) Shashi Tiwari
Assistant Professor
Shashi.tiwari@shiats.edu.in
Co- advisor Dr. Sunil Zacharia
Assistant Professor
sunil.zacharia@shiats.edu.in
Member Dr. (Mrs.) Sobita Simon
Professor & Head
Department of Plant Pathology and Entomology
sobita.devi@shiats.edu.in
Member Dr. (Mrs.) Anupriya Paul
Assistant Professor
Department of Mathematics and Statistics
anupriya.paul@shiats.edu.in
SAC Members

1. Introduction
2. Objectives
3. Material and Methods
4. Observations Recorded
5.Conclusion
6. References
CONTENTS

• Cabbage (Brassica oleracea L. var capitata) is one of the most important Cole crops belonging to
family Cruciferae and is grown for the thickened main bud called "Head". The word cabbage is an
anglicized form of the French "Cobbache" meaning head .
• . Cabbage heads generally range from 0.5 to 4 kilograms (1 to 9 lb.) and can be green. purple and white. It is a multi-
layered vegetable. Plants are 40-60 cm tall in their first year at the mature vegetative stage and 1.5-2.0 m tall when
flowering in the second year.
• Different varieties prefer different soil types ranging from lighter sand to heavier clay but all prefer fertile ground
with a pH between 6.0 and 6.8. Anonymous (2019-2020)
• China has the top position in cabbage production worldwide and India is on the 2nd position
• The Food and Agriculture Organization of the United Nations (FAO) reporting that total world production of all
Brassicas for 2019 was 70.104.972 metric tons
• The nations with the largest production were China which produced 47% of the world total and India which
produced 12%. China and India used a surface area of 980,000 hectares (2.400,000 acres) and 375,000 hectares
(930,000 acres) respectively.
INTRODUCTION

• Cabbage is a good source of vitamin K, vitamin C and dietary fiber. Cabbage provides 25kcal in form of
carbohydrate 5.8g, dietary fiber 2.5g. fat, 0.1g, protein 1.28g, vitamin B 0.671mg, vitamin C 36.6mg, vitamin
K 76µg. Calcium 40mg. Iron 0.47mg, Magnesium 12mg, Manganese 0.16 mg, Phosphorus 26 mg, Potassium
170 mg. Sodium 18 mg, Zinc 0.18 mg (Choudhury 1967).
• Such compounds include sulforaphane and other glycosylates which may stimulate the production of
detoxifying enzymes during metabolism. Studies suggest that cruciferous vegetables including cabbage may
have protective effects against colon cancer.
• The major cabbage producing states in India are Uttarakhand, Haryana, Rajasthan, Bihar, Gujarat,
Maharashtra, Orissa, Karnataka and Uttar Pradesh. The area, production and productivity of cabbage in India
were 403Mha,9369 Mt/ha, respectively [National Horticulture Board (2019-20)].
• For optimal growth there must be adequate levels of nitrogen in the soil especially during the early head
formation stage and sufficient phosphorus and potassium during the early stages of expansion of the outer
leaves
• . Temperatures between 4 and 24 °C (39 and 75 °F) prompt the best growth

• The cabbage crop is affected by various fungal as well as bacterial diseases like damping off, club rot, downy
mildew, Sclerotinia rot, black leg, black rot, soft rot and Alternaria blight or Alternaria leaf spot.
• The disease Alternaria leaf spot of cabbage is prevalent in all the cabbage growing states and is one of the
major biotic problems, which limits its production and also quality of produce.
• There are two species of Alternaria which cause serious damage in cabbage i.e., Alternaria brassicae and
alternaria hrassicicola.
• They can survive saprophytically outside of the host and diseased crop debris The primary sites of survival
from year to year are the resting spores (Yadav et al., 2014).
• At least 20% of agricultural spoilage is caused by Alternaria spp; most severe losses may reach up to 80% of
yield and 59% loss of cabbage seed yield may occur due to Alternaria blight (Hossain and Mian., 2005).
• Mycelial growth was most favored by 100% relative humidity with a gradual reduction in growth and
sporulation till 70% RH and a decrease in growth and sporulation at 60 and 50% RH (Meena et al., 2012).
• The genus Alternaria was first recognised by Nees in 1817. In 1836 Berkeley identified the causal fungus on
plants belonging to family Brassicaceae as Macrosporum brassicae (Berk), which was later renamed as
Alternaria brassicae (Berk) by Saccardo (1886)
• The most common symptoms of Alternaria brassicae are yellow dark brown to black circular spots with
target like concentric rings.

• Alternaria leaf spot/blight symptoms start as a small, circular, dark spot. As the disease progresses, the circular
spots may grow to ½ inch (1cm) or more in diameter and are usually gray, gray-tan, or near black in color.
• Spots develop in a target pattern of concentric rings. Dark, sunken lesions are usually the expressions of
Alternaria infections on roots, stems and fruits. The fungus may sporulate in these cankers, causing a fine,
black, velvety growth of fungus and spores to cover the affected area.
• Alternaria belongs to the sub-division Deuteromycotina, class Hyphomycetes, family Dematiaceae. Species of
the genus are cosmopolitan, surviving both as saprophytes as well as weak parasites. The genus is
characterized by the formation of polymorphous conidia either singly or in short or longer chains and
provided with cross, longitudinal as well as oblique septa and having longer or short beaks (Nayyar et al.,
2014).
• Plant essential oils are volatile compounds, broad spectrum, anti- fungal activity, eco-friendly and more
acceptable to the public. The mechanism of plant essential oils involves, inhibition of hyphal growth,
interruption in nutrient uptake, disruption of mitochondrial structure and eventually disorganization of fungal
pathogens discussed by (Patel and Jasrai, 2011).
• Use of eco-friendly alternative approaches for the management of plant diseases is the best for control of
fungal diseases because medicinal plants represent a rich source of antimicrobial agents (Mahesh and Satish,
2008).
• Many of these fungicides are bio-hazardous and adversely affect the components of ecosystem. Further, the
cost of these fungicides is comparatively high and their constant application results in development of
resistance in the pathogens against these fungicides (Nigam et al., 1994).

OBJECTIVES
1.To evaluate the effect of Trichoderma viride combination with selected essential oils on plant parameters of
cabbage in field conditions.
2.To evaluate the Disease intensity of Alternaria leaf spot disease of cabbage.

MATERIALS AND METHODS
The Present study on the “Efficacy of Trichoderma viride and selected botanical oils against Alternarial
leaf spot (Alternaria brassicae) disease of cabbage ” were carried out with a view to manage the Alternaria leaf
spot disease on cabbage with the help of botanical oils as seedling treatment was carried out during Zaid season
2020-2021 at the research field of Department of Plant Pathology, Sam Higginbottom University of Agriculture,
Technology and Sciences, Prayagraj. Field experiment was laid-out in Randomized block design with three
replications. The details of materials used and the methodology followed in conducting the experiments are
described in this chapter.

3.4. General laboratory procedures
3.4.1. Glassware cleaning:
3.4.2. Sterilization
3.4.3. Potato Dextrose Agar (PDA) media
Potato Dextrose Agar medium was used to isolate the pathogen Alternaria alternata from the diseased plant parts.
3.4.4. Isolation of Fungal Organism:
Potato Dextrose Agar (PDA) was prepared and 80 mg of streptomycin, an antibiotic was added to each 500 ml preparation
of the PDA at Luke warm PDA to inhibit probable bacteria growth. Diseased portion of the leaves is cut under aseptic conditions
into small bits into a sterile dish with the aid of scissors which was flamed over a spirit lamp flame and surface sterilized in
0.1 % mercuric chloride. The cut diseased and surface sterilized bits with 70% ethanol was placed on petri dishes poured with
solidified potato dextrose agar (PDA). The inoculated plates was incubated at room temperature until visible growths are seen on
the plates. The fungal colonies growing in the incubated plates was sub-cultured into fresh medium until pure cultures are
obtained.

3.5. Alternaria Leaf spot of cabbage
3.5.1. Symptoms
• Alternaria leaf spot/blight symptoms start as a small,
circular, dark spot. As the disease progresses, the
circular spots may grow to ½ inch (1cm) or more in
diameter and are usually gray, gray-tan, or near
black in color.
• Spots develop in a target pattern of concentric rings.
Dark, sunken lesions are usually the expressions of
Alternaria infections on roots, stems and fruits. The
fungus may sporulate in these cankers, causing a
fine, black, velvety growth of fungus and spores to
cover the affected area. Symptoms of Alternarial leaf spot of cabbage

3.5.2. Characteristics of pathogen
The morphological features of Alternaria brassicae were made
on host and in medium (PDA) by using compound microscope. On
the host, the pathogen produces concentric rings with greyish fungal
growth on the leaf. The spots produced are irregular in shape. In in
vitro conditions, the fungus grows fast producing colonies that are
black olivaceous black to greyish and appearance like suede to
floccose in Pertiplate. The mycelium is light brown to dark brown,
septate, branched. The conidiophore produces the conidia. Conidia
are muriform, ovate to beaked, they produce singly or in chains
(catenate).
Microscopic View of Alternaria brassicae

3.2. Experimental details:
3.2.1. Land preparation
The field was thoroughly ploughed and pulverized with tractor drawn cultivar to attain desirable tilt and one
harrowing. Levelling and formation of plots were done manually. The field was then cleaned by picking the
stubble of previous crop, all weeds and broken the soil clods with wooden hammer. The selected field was
divided into sub-plots (eighteen plots).

3.2.2. Transplanting
The experimental plot was laid out as per statistical design and
necessary marking of the hills was done for transplanting the seedling.
Proper care should be taken while selecting seedlings for transplanting.
Over and under aged seedlings should be avoided for better establishment.
The cabbage seedlings were transplanted by dibbling method. The seedlings
are treated with botanical oils and then they were transplanted on 1st
February 2021.
3.2.3. Gap filling
The dead or sick seedlings were replaced by healthy seedlings within a
week after transplanting. The damaged plants were also replaced by border
plants through gap filling.
Fig: An overview of Transplanting

3.2.4. Irrigation
Light irrigation was done after transplanting for the establishment of the crop and then subsequent irrigations was
done as per requirements. Irrigation was done after each weeding.
3.2.5. Weeding
Weeding was done when required to keep the crop free from weeds, for better soil aeration and conserve soil
moisture. The common weeds were Cynodon dactylon L. (Bermuda grass), Cyperus rotundus L. etc. Weeding was done
carefully keeping the delicate young plants undisturbed.
3.2.6. Plant protection measure
In order to protect the crop from insect pest and diseases standard methods of plant protection were followed whenever
needed.
3.2.7. Experimental design:
The experiment was laid out using Randomized Block Design (RBD) comprising of 6 treatments and 3
replications. The different treatments were allocated in each replication.

Experimental Design : Randomized Block Design (RBD)
Crop : Cabbage
Variety : golden acre
Number of Treatments : 06
Number of Replications : 03
Total No. of Plots : 18
Plot Size : 2 x 1 m
Net Plot size : 2 m2
Width of single plot : 2 m
Length of single plot : 1 m
Size of bunds : 0.3 m
Width of Irrigation channel : 1 m
Width of sub irrigation channel : 0.5 m
Row to Row Distance : 60 cm
Plant to plant distance : 45 cm
Total length of experimental area : 20.1 m
Total width of experimental area : 8 m
Gross cultivated area : 160.8 m2
Net cultivated area : 36 m2
Table No: Experimental details

T2 1 m T0
SUB-
IRRIGATION
CHANNRL
(0.5m)
T5
T0 2 m T4 T2
T5 T2 T4
T4 T5 T1
T3 T1 T3
T1 T3 T0
R1 R2 R3
9.1
m
Main Irrigation Channel
N
W E
S
Layout of Experimental field

Details of treatments
S. No
Treatments Treatment Details References
1) To Control (Untreated check) -
2) T1 Trichoderma viride@2.5% + Neem Seed kernel
extract@ 2.5%
Poonam et al. (2019)
3) T2 Trichoderma viride@2.5% + Clove oil@ 1.5% Ravinder et al. (2020)
4)
T3
Trichoderma viride@2.5% + Neem oil@ 2.5% Suryawanshi et al. (2019)
5) T4 Trichoderma viride@2.5% + Ginger oil 2.5% Mugao et al. (2020)
6) T5 Trichoderma viride@2.5% + Garlic
oil 2.5%
Manu Upadhyay et al. (2019)

3.2.8. Preparation and application of treatment
Using root dipping procedure
1.Trichoderma sp. was isolated from the soil of the experimental field with the help of serial dilution technique. Culture
proliferation was done with the help of single spore technique. Thereafter, with the help of scalpel 4 grams of mycelium
was taken and mixed with tale powder. The prepared powder formulation was then used for root dipping treatment.
2.As treatments was combination of Trichoderma viride and different essential oils, measured 2.5 gm of Trichoderma
viride powder to the 1000ml of water and dipped entire plant roots in that container for 30 min and transferred into
different essential oils containers.
Dipping in different essential oils:
1.Neem oil + Trichoderma viride:2.5ml of stock neem oil was measured using measuring cylinder and added a drop of
detergent to the 97.5ml of water in the conical flask to form 100ml of solution. Then the seedlings were dipped in the
treatment around 20-30 min and transplanted to plots.
2.Neem seed kernel extract + Trichoderma viride: 2.5ml of stock NSKE oil was measured using measuring cylinder
and added a drop of detergent to the 97.5ml of water in the conical flask to form 100ml of solution. Then the seedlings
were dipped in the treatment around 20-30 min and transplanted to plots.

3. Clove oil + Trichoderma viride: 1.5ml of stock clove oil was measured using measuring cylinder and added
a drop of detergent to the 97.5ml of water in the conical flask to form 100ml of solution. Then the seedlings
were dipped in the treatment around 20-30 min and transplanted to plots.
4. Ginger oil + Trichoderma viride: 2.5ml of stock ginger oil was measured using measuring cylinder and
added a drop of detergent to the 97.5ml of water in the conical flask to form 100ml of solution. Then the
seedlings were dipped in the treatment around 20-30 min and transplanted to plots.
5. Garlic oil + Trichoderma viride: 2.5ml of stock garlic oil was measured using measuring cylinder and
added a drop of detergent to the 97.5ml of water in the conical flask to form 100ml of solution. Then the
seedlings were dipped in the treatment around 20-30 min and transplanted to plots.

Measuring Trichoderma powder Measuring essential oils and dipping of seedlings

Observations recording Overview of plot

Leaf spot of cabbage after 22 DAT Infected plant healthy growing cabbage

3.3. Details of the observations recorded
3.3.1. Plant Growth Parameters
3.3.1.1. No. of Leaves at 30, 45, 60 DAT
3.3.1.2. Head Diameter
3.3.1.3. Head weight
3.3.2. Disease intensity (%)

3.3.2. Disease intensity
Disease intensity of plants was recorded at 30, 45, 60 DAT. In each plot, five plants were examined randomly
and scored for disease severity by following 0-5 scale as given by (Pandey et al., 2003).
Percent Disease Index (PDI) was calculated by using formula as given below Wheeler (1969).
Sum of numerical disease ratings
PDI = ------------------------------------------------------ X 100
No. of plants observed X Maximum disease rating

Grade Leaf area covered Reaction
0 No symptoms of disease on leaves. Immune
1 One or two necrotic spots on a few lower leaves
of plants, covering nearly 1-10% of the surface
area of the plant
Highly resistant
2 A few isolated spots on leaves, covering nearly
11-25% of the surface area of the plant
Resistant
3 Many spots coalesced on the leaves, covering 26-
50% of the surface area of the plant
Moderately resistant
4 Irregular, blighted leaves and sunken lesion with
prominent concentric rings on the stem petiole,
fruit, covering 51-75% leaf area of the plant.
Moderately susceptible
5 Whole plants blighted, leaf and fruits starting to
fall, covering more than 75% leaf area of plant.
Highly susceptible
Disease Scale Grade

RESULTS
The results obtained in the study entitled “Efficacy of Trichoderma viride and selected botanical oils
against Alternarial leaf spot (Alternaria brassicae) disease of cabbage ” are presented and discussed below.
Table no. 4.1 Effect of Essential oils on No. of Leaves at 30, 45 and 60 DAT of Cabbage.
SN. No Treatme
nt No.
Treatment name No. of leaves
30 DAT 45 DAT 60 DAT
1
T0 Control (untreated) 8.66 14.33 24.75
2
T1 Trichoderma viride@2.5% + NSKE@ 2.5% 10.26 15.26 28.66
3
T2 Trichoderma viride@2.5% + Clove oil@ 1.5% 11.66 18.75 28.00
4
T3 Trichoderma viride@2.5% + Neem oil@ 2.5% 15.75 27.66 36.75
5
T4
Trichoderma viride@2.5% + Ginger oil @2.5%
13.73 22.36 32.75
6
T5 Trichoderma viride@2.5% + Garlic oil@ 2.5% 10.23 27.66 34.4
F-Test
S S S
CD (5%) 1.07 1.58 1.28
S.Em ± 0.43 0.79 0.57

Figure No. 4.1 Effect of Essential oils on No. of Leaves at 30, 45 and 60 DAT of cabbage
0
5
10
15
20
25
Control (untreated) Trichoderma
viride@2.5% +
NSKE@ 2.5%
Trichoderma
viride@2.5% + Clove
oil@ 1.5%
Trichoderma
viride@2.5% +
Neem oil@ 2.5%
Trichoderma
viride@2.5% +
Ginger oil @2.5%
Trichoderma
viride@2.5% +
Garlic oil@ 2.5%
sNo.
of
Leaves
Treatments

Table no. 4.5 Effect of Essential oils on Leaf spot Disease intensity of alternarial leaf spot of cabbage at
different days of intervals.
Treatments
PDI (%)
T0 Control 28.69 38.75 59.66
T1
Trichoderma viride + Neem oil @
2.5%
21.54 24.32 30.66
T2 Trichoderma viride + NSKE @ 2.5% 23.66 38.75 40.33
T3
Trichoderma viride + Clove oil @
2.5%
19..33 22.75 28.65
T4
Trichoderma viride + Garlic oil @
2.5%
18.65 24.69 34.33
T5
Trichoderma viride + Ginger oil @
2.5%
20.65 31.23 32.45
Result
S S S
C.D at 5 %
0.07 1.04 0.96
0.45 0.48 0.45

Figure no. 4.2 Effect of Essential oils on Leaf spot Disease intensity of Cabbage
at different days of intervals.
0
10
20
30
40
50
60
viride@2.5% +
NSKE@ 2.5%
Trichoderma
viride@2.5% +
Clove oil@ 1.5%
Trichoderma
viride@2.5% +
Neem oil@ 2.5%
Trichoderma
viride@2.5% +
Ginger oil @2.5%
Trichoderma
viride@2.5% +
Garlic oil@ 2.5%
Disease
intensity(%)
Treatments

SN. No Treatm
ent No.
Treatment name head weight ( gram)
1
T0 Control (untreated) 100 240 480
2
T1 Trichoderma viride@2.5% + NSKE@ 2.5% 200 280 560
3
T2 Trichoderma viride@2.5% + Clove oil@ 1.5% 220 340 645
4
T3 Trichoderma viride@2.5% + Neem oil@ 2.5% 250 475 850
5
T4
Trichoderma viride@2.5% + Ginger oil @2.5%
230 360 660
6
T5 Trichoderma viride@2.5% + Garlic oil@ 2.5% 240 430 670
F-Test
S S S
CD (5%) 1.07 1.58 1.75
S.Em ± 0.83
Table no. 4.3 Effect of Essential oils on head weight of cabbage at different days of intervals.

0
100
200
300
400
500
600
700
800
900
viride@2.5% +
NSKE@ 2.5%
Trichoderma
viride@2.5% + Clove
oil@ 1.5%
Trichoderma
viride@2.5% + Neem
oil@ 2.5%
Trichoderma
viride@2.5% + Ginger
oil @2.5%
Trichoderma
viride@2.5% + Garlic
oil@ 2.5%
chart no. 4.3 Effect of Essential oils on head weight of cabbage at different days of intervals.

• Cabbage (Brassica oleracea L. var capitata) is one of the most important Cole crops belonging to family
Cruciferae and is grown for the thickened main bud called "Head". Cabbage is a good source of vitamin K.
vitamin C and dietary fiber.
• The cabbage crop is affected by various diseases like damping off, club rot, downy mildew, Sclerotinia rot,
black leg, black rot, soft rot and Alternaria blight or Alternaria leaf spot. Among these diseases Alternaria leaf
spot of cabbage is prevalent in all the cabbage growing states and is one of the major biotic problems, which
limits its production and also quality of produce
• There are two species of Alternaria which cause serious damage in cabbage i.e.., Alternaria brassicae and
Alternaria hrassicicola
• Keeping in view the present study of the research entitled “Efficacy of Trichoderma viride and selected
essential oils against Alternarial leaf spot (Alternaria brassicae) disease of cabbage” under field condition
was conducted at the Department of Plant Pathology, Sam Higginbottom University of Agriculture,
Technology and Sciences, Prayagraj, during Zaid season 2021, with the following objectives
• 1.To evaluate the efficacy of Trichoderma viride combination with different essential oils against
Alternaria leaf spot of cabbage in field conditions.
• 2.To evaluate the Disease intensity of Alternaria leaf spot of cabbage

• The field experiment was analyzed by using R.B.D. (Randomized block design) with three replications in a
plot size 2x 1m2 and six treatments like control (untreated check), T1 root dipping treatment of Trichoderma
viride@2.5% + Neem Seed kernel extract@ 2.5 ml/lit, T2 root dipping treatment of Trichoderma viride@2.5%
+ Clove oil @ 2.5 ml/lit, T3 root dipping treatment of Trichoderma viride@2.5% + Neem oil @ 2.5 ml/lit, T4
root treatment of Trichoderma viride@2.5% + Ginger oil @ 2.5 ml/lit and T5 root treatment of Trichoderma
viride@2.5% + Garlic oil @ 2.5 ml/lit.
• The maximum number of leaves @30 DAT of cabbage was significantly increased in T3 root dipping
treatment of Trichoderma viride @2.5% + Neem oil @ 2.5 ml/lit followed by T1 root dipping treatment of
Trichoderma viride @2.5% + Neem Seed Kernal Extract @2.5 ml/lit compared to control.
• Results showed that minimum disease intensity@30 DAT of cabbage was significantly increased in T3 root
dipping treatment of Trichoderma viride @2.5% + Neem oil @ 2.5 ml/lit followed by T1 root dipping
treatment of Trichoderma viride @2.5% + Neem Seed Kernal Extract @2.5 ml/lit compared to control .

CONCLUSION
• Alternaria leaf spot is a destructive disease that causes higher yield losses in cabbage as there is direct
association of this disease in head weight and yield loss. Hence the management of this disease is important.
The present study revealed that Trichoderma viride in combination with neem oil @ 2.5 ml/lit as root
treatment was most effective against the disease in minimizing the disease intensity and enhance the plant
growth parameters of crop compared to other treatments. Thus, the present study indicated that suitable
integration of more efficient eco-friendly treatments like bio-agents and essential oils may provide a better
and effective management of Alternaria leaf spot disease of cabbage, but there is a scope for more
experimental trails to further validate the results on larger basis. The findings of the present experiment are
limited to one crop season under Prayagraj agro-climatic conditions, as such to validate the present findings
more such trails should be carried out in future

BIBILIOGRAPHY
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leaf blight of tomato in Pakistan, Plant Pathology, 53 (6):816.
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• Chandan, K.S., Samir, P.S., Tiwari, N.K., Triveni, T. and Dharmendar, S. (2015). Bio-Efficiency of plant extract,
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• Khodke, S.W, Pawar, R.V. and Bhopale, A.A. (2000). Pathogencity of Alternaria alternata (Fr.)
Keissler causing leaf spot disease of chilli, PKV Research Journal, 24 (2): 123.
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