SYNOPSIS

On
Evaluation of oilcakes and phyto-extracts for the management of
Alternaria leaf spot on Bok-choy (Brassica chinensis L. )

ADVISOR: PRESENTED BY:

Dr. (Mrs.) Abhilasha A. Lal Reveshi Sudhanshu
Assistant Professor PID No. 22MSAPP100
Department of Plant Pathology M. Sc. (Agri.) Plant Pathology
NAI, SHUATS 4th Semester

DEPARTMENT OF PLANT PATHOLOGY

NAINI AGRICULTURAL INSTITUE

SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE,

TECHNOLOGY AND SCIENCES

PRAYAGRAJ (U.P.)

1
 INTRODUCTION

Bok choy is a type of Chinese cabbage (Brassica rapa or Brassica campestris) of the chinese
group, Sometimes called Brassica chinensis. It is a loose leaf, non-heading type of chinese
cabbage with thick white leafstalks with green leaves that form in clusters. Cultivated since
the 5th century in Asia, bok choy is now grown in many countries around the world
(Stephens, 2015).

There are many cultivars/varieties of bok choy, and many common names including pai-tsai,
pak choi, pe-tsai, and more. Some common names of bok choy overlap with common names
of the other type of chinese cabbage, brassica rapa pekenensis group. Varieties of B. rapa
pekenensis group, sometimes called b. pekenensis, are broadleaved, compact-heading type
Chinese cabbages (Stephens, 2015).

Bok choy is most widely consumed as a fresh leafy vegetable in dishes like salads and the
popular Korean kimchi dish.

Bok choy is low in saturated fat and cholesterol. It is also a good source of dietary fiber,
protein, thiamin, niacin, and phosphorus, and a very good source of dietary fiber, vitamin A,
vitamin C, vitamin K, vitamin B6, folate, potassium and manganese (Self Nutrition Data,
2014).

Bok choy is considered a cool-season crop that can withstand light frost. It grows best in
temperatures of 55 to 70°F (13-21°C), but can tolerate higher temperatures with ample soil
moisture (Tuquero et al.,2018 ).

Cultivation of bok choy is done mainly on sandy to heavy soils rich in organic matter. Early
crops prefer light soil while late crops thrive better on heavier soils due to retention of
moisture. On heavy soils, plants grow more slowly and the keeping quality is improved. A
pH range of 5.8-7.5is considered as optimum for growing bok choy. Plants growing in saline
soils are prone to diseases. In India, bok choy is grown in large areas having a cool and moist
climate. The intensity of flowering depends upon the age of the plants and the period for
which they are exposed to low temperatures (NHB,2018-2019).

It prefers winter temperature for optimum growth and yield, although today offseason
cultivation is also increasing with the introduction of suitable varieties. India ranks second

2
after China for bok choy production with 48% of world production (Sharma et al., 2017). In
India, it is grown in 3.98 lakh hectares with a production of 90.37 lakh tonnes and a
productivity of 22.70 tonnes per hectare (NHB, 2018). The major cabbage producing states
are Arunachal Pradesh, Assam, Meghalaya, Manipur, Mizoram, Nagaland and Tripura
(Majid, 2020).

Bok choy is a host for a wide range of pests and diseases. Some common diseases that infect
bok choy include Black Rot (Xanthomonas campestris) (bacterium), Powdery Mildew
(Sphaerotheca spp.) (fungus), and leaf spot (Alternaria brassicae) (fungus). Some common
pests that infest bok choy include cabbage webworm (Hellula undalis), diamondback moth
(Plutella xylostella), aphids (Family: Aphididae), cabbage looper (Trichoplusia ni), and red
fire ants (Family: Formicidae) (Kelley, 1999).

Alternaria black leaf spot disease is one of the most common and destructive diseases of
cabbage and brassicas worldwide (Meah et al., 2002). It usually occurs during warm and
moist weather. A complex of Alternaria species (A. brassicicola (Schw.) Wiltsh., A.
brassicae (Berk.) Sacc., A. alternata (Fr.) Kreissler and A. raphani Groves and Skolko) is
responsible for important yield losses (Verma and Saharan, 1994) causing reduction of
photosynthetic potential, accelerating senescence, defoliation, premature pod shatter and
shriveled seed leading to considerable reduction in quality and quantity of yield products
(Shresta et al., 2000 and Dharmendra et al., 2014).

The causal agent of the leaf spot disease of cabbage is known to be Alternaria brassicicola.
The infected leaves appear dark brown to black, zonate spots are circular (1 to 10 mm in
diameter), in culture the colonies look amphigenous dark olivaceous brown. The mycelium is
septate, hyphae branched, hyaline to brown, inter and intracellular. Conidiophores arise
singly or in groups. The conidia are in chains, some times branched, arise through small pores
from the conidophore wall, straight, tapering towards the apex, the basal cell rounded, the
beak present or absent, apical cell more or less rectangular, contain less than 6 transverses
septa and few longitudinal septa. Conidia are constricted at the septa (Ellis, 1971 and Singh,
1982).

3
 JUSTIFICATION

For the management of pathogens for Bok-choy crop from many years, many have been
relied on chemicals and this resulted in many undesirable problems. There is need to
incorporate alternative control components that are effective in field and are organically
developed for growing concern of both public health and environment hazards thus less
costly and without affecting public health and environment. Considering the mentioned facts,
a study entitled, “Evaluation of oilcakes and phyto-extracts for the management of
Alternaria leaf spot on Bok-choy (Brassica chinensis L. ) ”is proposed with the following
objectives.

OBJECTIVES

1. To evaluate the effect of treatments on the plant growth parameters of Bok-choy under
field condition.
2. To evaluate the effect of treatments on Disease Intensity in Bok-choy.
3. To calculate Cost Benefit Ratio of the treatments.

4
 REVIEW OF LITERARUTE

Sasode et al. (2012) reported that all the tested botanicals were found effective against
Alternaria brassicae. Among the crude extract 10 per cent the minimum growth was recorded
in Neem followed by eucalyptus, tulsi, lantana, datura and pudina. Neem was significantly
superior over tulsi, lantana, datura and pudina but at par with eucalyptus. Under boil forms
the minimum radial growth was also recorded in neem. The oil extract (neem and eucalyptus)
were found less effective as compared to crude and boil extracts. Neem crude extract is
confirmed to have antifungal effect on Alternaria brassicae.

Kabir et al. (2014) an experiment was conducted to find out the effect of some organic foliar
treatments to manage the Alternaria leaf blight disease in broccoli (cv. Green Sprouting) was
studied under natural field condition. Besides field sanitation, aqueous extracts of Neem,
Mahogany (Swietania mahagoni), Koromcha (Carissa carandas) and Garlic clove (Allium
sativum) administered as foliar spray. All the treatments controlled the Alternaria blight
disease of broccoli significantly giving higher yield compared to control.

5
Sadana et al. (2015) study revealed that all the seven fungicides (mancozeb, captan, thiram,
coppersulphate, carbendazim, zineb and copper oxychloride) reduce the disease severity as
compared to untreated check. The highest reduction in the disease was achieved by applying
mancozeb (1500 ppm) that caused 86.4 percent inhibition of mycelial growth in A1 strain of
Alternaria solani. Bio-efficacy of fifteen plant extracts (Polyalthia longifolia, Azadirachta
indica, Datura stramonium, Ocimum sanctum, Calotropis procera, Crotalaria juncea,
Eucalyptus obliqua, Cassia fistula, Agele marmelos, Croton bonplonadium, Pergularia
daemia, Cleome viscose, Phyllanthus amarus, Bauhinia purpurea, Euphorbia hirta) were
evaluated under in vitro conditions. Among plant extracts evaluated, fresh aqueous extract of
Eucalyptus obliqua (15%) was effective in causing 88 percent inhibition of mycelial growth
in A1 strain of Alternaria solani. followed by Datura stamonium, Azadirachta indica,
Calotropis procera and Polyalthialongifolia. Thus plant extracts have shown significant
inhibition and proved to be cost effective and ecofriendly for the management of Alternaria
solani and were comparable with fungicides.

Singh et al. (2015) conducted an experiment and found that foliar spray of mancozeb (T5)
was effective as compared with other treatments, seed treatment and foliar spray of
Trichoderma viride (T6 ) was effective in reducing alternaria leaf spot followed by seed
treatment and foliar spray of garlic bulb extract (T3 ), seed treatment and foliar spray neem
leaf extract (T1 ), seed treatment and foliar spray of onion bulb extract (T4 ) and foliar spray
of black nightshade leaf extract (T2 ) under Allahabad conditions. The maximum fresh
weight of plant, diameter of cabbage bud, weight of cabbage head and yield of cabbage were
maximum in treatments T5 followed by T6 , T3 , T1 , T4 and T2 , however, the germination
per cent was maximum in plot treated with T5 followed by T3 , T1 , T4 , T2 and T6 as
compared to control (untreated plot).

Patel et al. (2018) study was conducted for management of cauliflower in which several
fungicides have used against Alternaria brassicae and few are highly effective. Therefore,
present investigation was conducted on efficacy of different fungicides against Alternaria
brassicae caused Alternaria leaf spot of cauliflower. Five fungicides viz. Azoxystrobin
(Onestar 23% SC), Carbendazim (Dhanustin 50%WP), Kasugamycin (Kasu B 3% SL),
Fusilazole (Cursor 40% EC) and Pyraclostrobin + Metaram (Carbrio Top 60% WG), were
assayed at two concentration 300ppm and 550ppm for their efficacy against A. brassicae.

6
Biswas and Ghosh (2018) conducted an experiment on Alternaria leaf blight caused by
Alternaria brassicae and A. brassicola, one of the most destructive diseases of mustard
(Brassica campestris, B.juncea and B.napus) causing considerable damages to the crop under
in vitro and in vivo conditions to see the effect of bio-agents, plant extracts and chemicals
(fungicide and SAR compound) against A. brassicae. Maximum inhibition in mycelial
growth (95.56%) was observed with Mancozeb 75% Wp followed by Lantana camara
(80%), Salicylic acid (73.33%), Allium sativum(54.44%) and Zingiber officinale (17.78%) in
comparison to control. Foliar spray with fungicide (Mancozeb 75% WP @ 0.2%) was found
to be most effective in reducing disease severity (81,23%) and infection rate which increased
the yield (77.23%) of mustard over untreated control. Among the plant extracts, Lantana
camara was found to be excellent in controlling the alternaria blight infection in the field
(71.92% reduction in disease severity and 68.18% increase in yield) in comparison to
salicylic acid (SAR compound) and bio-agent (Trichoderma viride) 48.33% and 36.27%
reduction in disease severity respectively.

Sreevarshini et al. (2019) investigated an experiment and found that the minimum disease
intensity was recorded in foliar sprays of Eucalyptus globulus at 10% (30.40 %) followed by
Azadirachta indica at 15% (31.4 %), Lantana camara at 15% (32.40 %), Ocimum sanctum at
15% (34.8 %), Cyperus rotundus at 15 % (37.4 %), as compared to treated (27.5 %) and
untreated (43.8 %) checks. Maximum fresh head weight was recorded in Eucalyptus globulus
untreat (0.80 kg ) followed by Azadirachta indica (0.65 kg ), Lantana camara (0.64 kg ),
Ocimum sanctum (0.56 kg ), Cyperus rotundus (0.52 kg ), as compared to treated (1.01 kg)
and untreated (0.46 kg) checks. Maximum yield was recorded in Eucalyptus globulus (26.72
tonnes/ha) followed by Azadirachta indica (24.44 tonnes/ha), Lantana camara (22.88
tonnes/ha), Ocimum sanctum (21.27 tonnes/ha), Cyperus rotundus (17.77 tonnes/ha), as
compared to treated (33.96 tonnes/ha) and untreated (14.71 tonnes/ha) checks.

Sreevarshini et al. (2019) experimented for management practices on leaf spot of cabbage
using botanicals was conducted during Rabi season. The minimum disease intensity was
recorded in foliar sprays of Eucalyptus globulus @ 10% (30.40%) followed by Azadirachta
indica @ 15% (31.4%), Lantana camara @ 15% (32.40 %), Ocimum sanctum @ 15%
(34.8%), Cyperus rotundus @15 % (37.4%), as compared to treated (27.5%) and untreated
(43.8%) checks.

7
Solanke et al. (2020) evaluated that organic manure significantly reduced the alternaria leaf
spot of Cabbage (Alternaria brassicae) where among the organic manure amendments soil
application farm yard manure at 6.0 tonns/ha increased the yield. The maximum cost benefit
ratio was recorded by farm yard manure 6.0 tons/ha. It also concluded that Neem cake at 500
kg/ ha were found most effective in managing the alternaria leaf spot of cabbage with better
yield.

Dhaka et al., (2022) assessed the efficacy of nine phytoextracts and four oil cakes against the
pathogen Alternaria solani, causing early blight of tomatoes. Among the phytoextracts,
Azadirachta indica at 30% concentration was most effective and inhibited mycelial growth
by 74.67%, followed by Pongamia pinnata (65.64%), Ocimum sanctum (57.74%),
Eucalyptus globulus (56.53%), and Jatropha curcas (54.64%) after 120 h of inoculation. In
the case of oil cakes, maximum inhibition (51.51%) was recorded after neem cake-based
preparations, followed by mustard (44.56%) and cotton (42.75%). Minimum inhibition of
lineseed (34.48%) after 120h was recorded when linseed cake-based product was used.

Patel et al., (2023) an experiment was conducted for knowing the potentiality of different
seedling growing media viz., Cocopeat: Perlite: Vermiculite.

MATERIALS AND METHOD

8
The present study entitled “Evaluation of oilcakes and phyto-extracts for the
management of Alternaria leaf spot on Bok-choy (Brassica chinensis L. ).” will be
conducted at Central Research Field of Sam Higginbottom University of Agriculture,
Technology and Sciences, Prayagraj during the Rabi season of 2023. The details of materials
which will be used and the method which will be adapted in carrying out the studies are
presented below.

Field experiment

Field preparation: The selected field area will be well prepared and plot marked as per the
layout plan. The selected field will be dug up, weeded, cleaned and the soil will be pulverized
after which the total area divided into sub-plots.

Location: The research farm is situated on the right side of Central Research Field, Sam
Higginbottom University of Agriculture Technology and Sciences, Prayagraj. The site which
will be selected is uniform, cultivable with typical black soil having good drainage.

Climate and weather conditions: Prayagraj is situated at 25⁰ 27 ‫ ׳‬North latitudes and 80⁰

50 ‫ ׳‬East longitudes and at an altitude of 98 meter above sea level. Prayagraj region has sub-
tropical and semi-arid climate with the monsoon commencing from July and with drawing by
the end of September. The temperature goes up to 47 ⁰C during summers and goes down to
2.5⁰C in winter.

General Laboratory Procedures

Glassware cleaning: For all laboratory experiments, Borosil and Corning glassware will be
used. The glass wares were kept for 24 hours in the cleaning solution containing 60.0 g of
potassium dichromate, 60.0 ml of concentrated sulphuric acid in 1000 ml of water. They will
be washed with detergent solution followed by rinsing with tap water and finally with
distilled water.

Sterilization: The Petri plates and pipettes will be wrapped in clean paper and sterilized in
hot air oven at a temperature of 160°C for two hours. Sterilization of both solid and liquid
media will be achieved by autoclaving at 15lbs (121.6°C) pressure for 20 minutes for all the
laboratory studies. All cultural studies will be conducted in aseptic condition under laminar
airflow. The tips of inoculation needle, forceps & cork borers will be sterilized under flame.
9
Potato Dextrose Agar (PDA) Media: Potato Dextrose Agar medium will be used to isolate
the pathogen Alternaria sp. from the Diseased plant parts (Shadwick, 1938).

The composition of PDA is as follows;

1) Peeled potato tuber : 200 g

2) Dextrose : 20g
3) Agar : 20g
4) Distilled water : 1000 ml
5) pH : 6.0 - 6.5

Procedure:

The potato will be peeled and cut in to small pieces and boiled in 500 ml of distilled water till
they become soft. The extract obtained will be filtered through muslin cloth and all the liquid
will be squeezed in beaker. 20g of dextrose will be added bit by bit to the remaining 500 ml
of hot water. Then 20 g of agar will be added to solidify. Volume of broth will be made up to
1000 ml by adding more distilled water. Then in each conical flask 200 ml of this solution
will be dispensed and sterilized at 121°C at 15 lbs. pressure/square inch for 15 minutes in an
autoclave.

Slant preparation and PDA plating:

Potato Dextrose Agar media after preparation is cooled down to 45°C and poured into each
test tube about 5-6 ml, of 10ml capacity. Cotton plugs made up of non-absorbent cotton will
be used for plugging the culture tube. The test tubes will be sterilized in an autoclave by
routine method. After sterilization test tubes were slanted by putting in a wooden slanter.
After solidification, they were used for inoculating the pathogen and preparing tube culture.
Sterilized solid media will be cooled down at 40°C and 15-30ml will poured in each
sterilized petri-plate of 90mm diameter. After solidification, they were used for inoculating
the pathogen and preparing culture plates.

Isolation of fungal organism:

Initially the diseased leaves will be collected from infected plants and thoroughly washed
under running tap water. The diseased portion of the leaves will be cut under aseptic

10
conditions into small bits and surface sterilized with 0.1% NaOCl solution for 1 minute and
will be washed three times with sterile distilled water and placed on media. To avoid bacterial
contamination streptomycin @ 100 ppm, will be added in the medium at lukewarm stage
before pouring PDA into Petri plates. Then Petri plates will be wrapped and incubated at
27±2°C in BOD. To get the pure culture of the fungus, hyphal tip method will be used for
sub-culturing the fungus in media slants/petri-plates. The culture will be periodically
transferred to fresh media (Thilagam et al., 2017) .

Identification of the pathogen:

Morphological studies of the pathogen will be conducted from infected leaf by placing the
half-infected portion and healthy portion on a slide, later stained using lacto phenol and
cotton blue and morphological characters were noted with help of microscope. And using a
sterile needle, a small portion of the culture is taken and placed on a sterile glass slide,
stained using lacto phenol and cotton blue. Then the microscope is used for the examination
of morphology and culture characteristics of fungi (Barnett and Hunter 1973).

Identification of the fungus by slide preparation:

Examination of fungal colony characteristics will be done through microscope examination.

Materials required:

1. Disease sample .
2. Glass slide .
3. Cover slip .
4. Lacto phenol .
5. Cotton blue .
6. Needle .
7. Forceps .
8. Compound microscope .

Method:

11
1. Using a sterile needle, a small portion of the culture will be taken and placed on a sterile
glass slide.
2. It will be stained using lacto phenol and cotton blue and cover it with the cover slip.
3. Then, the microscope will be used for the examination of morphological characteristics of
fungal structures. (Grahovac et al., 2012).

Characteristics of the pathogen:

1. The first symptom of the disease appears with minute yellow specks on mature leaves and
stems.
2. The irregular dark-brown to black necrotic spots of variable size on the surface of the
leaves can be seen.
3. The hyphae were septate and dark brown in colour.
4. The conidia were 22 to 71 μm long and 9 to 16 μm wide, straight to ovoid, occasionally
tapered toward their apex, and formed in long chains, dark to olive brown in colour, with
four to seven transverse septa and zero to three longitudinal septa.
5. The conidia either had small to large beaks.
6. The conidiophores were both solitary and in groups and were straight to flexuous.
(Nira et al.,
2022)

Scientific Classification:

Kingdom - Fungi

Phylum - Deuteromycota

Class - Hyphomycetes

Order - Moniliales

Family - Dematiaceae

Genus - Alternaria

Species - brassicae, brassicicola (Ainsworth 1973).

12
 PLAN OF WORK

The field experiment will be conducted at the research plot of the Central Research Field,
Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj during the
Rabi season of 2023-2024.

Experimental Details:The experimental details are given below under different headings.

Details of the crop:

Name of the selected crop : Bok choy

Crop spacing : 45x30 cm

Duration : 40-60 days

Season : Rabi Season

Experimental Design:

Randomized Block Design (R.B.D) comprising of 7 treatments and 3 replications.

Details of Experiment:

Experimental design : Random Block Design (RBD)

Number of replications : 03

Number of treatments : 07

Total number of plots : 21

Plot size : 2.0 x 1.0 = 2.0 m2

Size of bunds : 0.3m

Width of main irrigation channel : 1m

Width of sub-irrigation channel : 0.5m

Total gross cultivated area : 89.8m2

Total net cultivated area : 48m2

Spacing Row to Row : 45-60 cm

13
Plant to plant : 30-45 cm

LAYOUT OF EXPERIMENTAL FIELD

14
S. No. Treatments Treatment Details References

1. T0 Control (Untreated) --

2. T1 Neem cake 40g/m2 (S.T) Sreevarshini et al. (2019)

+Eucalyptus globulus @10% (F.S)

3. T2 Linseed cake 40g/m2 (S.T) +Neem Choudhary et al. (2020)

leaf extract (Azadirachta indica) @
10% (F.S)

4. T3 Mustard cake 40g/m2 (S.T) + Yadav et al. (2014)

Lantana camara @10% (F.S)

5. T4 Neem cake 20g/m2 + Linseed cake Gupta et al. (2019)

20g/m2(S.T) + Calotropis giganta
leaf extract @10% (F.S)

6. T5 Neem cake 20g/m2 + Mustard cake Tijjani et al. (2014)

20g/m2(S.T) +Moringa stenopetala
@10%(F.S)

7. T6 Neem cake 20g/m2 + Mustard cake Biswas and Ghosh (2018)

20g/m2 + Linseed 20g/m2 (S.T) +
Mancozeb (Treated Check)
TREATMENT DETAILS IN FIELD CONDITION

*S.T. – Soil Treatment

15
*F.S. – Foliar Spray

OBSERVATIONS TO BE RECORDED

Observations under field condition

 Plant height (cm) .

 No. of leaves per plant .
 Average leaf length per plant .
 Disease severity.
 Yeild (t/ha).

Disease Measurement

It is a measurement of prevalence of the disease in the population and reflects the number or
proportion of plants units diseased. In measurement of disease the three assessment that are
commonly employed are (a) the incidence that is the number of individuals showing the
disease, (b) the severity of the disease, that is the proportion of area or amount of plant tissue
that is diseased, and (c) the loss caused by the disease. In many other diseases such as most
leaf spot it has little relationship to the severity of the disease which is expressed as the
percentage or population of plant area destroyed by the pathogen. It is closer to the yield loss
caused by the disease. The yield loss is positively correlated with economic loss. As it is leaf
spot diseases so its effect should be measured by counting no. of affected leaves among
healthy ones.

Description of Disease intensity was calculated by applying 1-5 grade disease rating scale for
Alternaria leaf spot of broccoli (Sangeetha and Siddaramaiah, 2007).

Class/Grade Description

0 No infection.

1 <5% infection.

2 5-10% infection.

3 10-25% infection.

4 25-50% infection.

16
 5 >50% infection.

Disease Intensity

It is calculated by using the following formula (Wheeler, 1969).

Disease Intensity ( % )=
∑ of all disease ratings x 100
Total number of rating x Maximum disease grade

Cost Benefit Ratio

Gross returns will be calculated by multiplying total yield with market price of the produce.
Cost of cultivation and cost of treatments will be deducted from the gross returns, to find out
returns and cost benefit of ratio by following formula.(Reddy, Reddi 2004)

The B:C ratio will be calculated by the given formula:

Gross return
Benefit Cost Ratio=
Total cost of treatment

17
 STATISTICAL ANALYSIS

In the experiment, Randomized Block Design (RBD) is to be adopted. The analysis of

variance
(ANOVA) technique will be applied for drawing conclusion from data. The calculated values
will be compared with the tabulated values at 5% level of probability for the appropriate
degree
of freedom (Fisher and Yates, 1964).The skeleton of analysis at variance table is as follows:
Analysis of variance table (ANOVA):

F(tab.)
Source of
D.f S.S M.S.S F(cal.) at Result
variation
5%

Due to M.S.S.R
replicatio r-1 R.S.S = M.S.S.R/M.S.S.E F(r-1) S/NS
n R.S.S/r-1
Due to M.T.S.R
F(r-1)
treatment t-1 T.S.S = M.T.S.S/M.S.S.E N/NS
(t-1)
s T.S.S/t-1

M.S.S.E f (t-1),
Due to
(r-1) (t-1) E.S.S = - (r-1), (t- -
error
S.S.E/(r-1) (t-1) 1)

Total (rt-1) T.S.S - - - -

S.E (d) for treatment = √(2 × MESS)/𝛾

C.D. at 5% = S.E (d) x t(5%) ed

Where,
D. f. = Degree of freedom
S. S. = Sum of square

18
M.S.S. = Mean sum of square
F(cal.) = Calculated value of ‘F’
F(tab.) = Table value of ‘F’
R. S. S. = Sum of square due to replicate
E. S. S. = Error sum of squares
T. S. S. = Total sum of squares
M. S. S. R. = Mean sum of squares (Replication)
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