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Integrated Disease Management against Fusarium oxysporum f.sp. lycopersici

on Tomato (Lycopersicon esculentum L.)
Thesis · September 2015
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Integrated Disease Management against Fusarium oxysporum
f.sp. lycopersici on Tomato (Lycopersicon esculentum L.)
Ph.D. THESIS
Submitted in partial fulfillment of the requirements for the award of the degree of
DOCTOR OF PHILOSOPHY
IN
PLANT PATHOLOGY
BY
HUSSEIN ALI SALIM
ID. No. 12PHCPP103
FACULTY OF AGRICULTURE, SCHOOL OF AGRICULTURE

DEPARTMENT OF PLANT PATHOLOGY
SAM HIGGINBOTTOM INSTITUTE OF AGRICULTURE,
TECHNOLOGY AND SCIENCES
Deemed-to-be-University
) FORMERLY ALLAHABAD AGRICULTURAL INSTITUTE)
NAINI, ALLAHABAD – 211007, INDIA
2015
CERTIFICATE OF ORGINAL WORK
It is certified that the thesis entitled ‘‘Integrated Disease Management against

Fusarium oxysporum f.sp. lycopersici on Tomato (Lycopersicon esculentum L.)”
submitted to Sam Higginbottom Institute of Agriculture, Technology and Sciences,
Deemed University, Naini, Allahabad in partial fulfillment of the requirement for the
degree of Doctor of Philosophy in Plant Pathology Faculty of Agriculture is a
bonafide record of research carried out by (Mr. HUSSEIN ALI SALIM and ID No:
12PHCPP103) under my supervision and guidance. The thesis is recommended for
acceptance.
Place: Allahabad Prof. (Dr.) Sobita Simon

Date: Advisor:
Department of Plant Pathology
THE CERTIFICATE OF ACCEPTANCE OF EVALUATION COMMITTEE
This thesis entitled ‘‘Integrated Disease Management against Fusarium oxysporum

f.sp. lycopersici on Tomato (Lycopersicon esculentum L.)” , has been prepared and
submitted by Mr. HUSSEIN ALI SALIM Id. No. 12PHCPP103 in partial fulfillment
of the requirements for the award of degree Doctor of philosophy in PATHOLOGY,
Allahabad School of Agriculture, Faculty of Agriculture, Sam Higginbottom Institute of
Agriculture, Technology and Sciences, Allahabad-211007, Uttar Pradesh, and is hereby
accepted for the award of the degree.
Name Evaluation Signature

1. Dr. (Mrs.) Sobita Simon
Professor
Department of Plant Pathology Satisfactory/
(Advisor) Not satisfactory ---------------
2. Dr. Abhilasha A. Lal

Assistant Professor Satisfactory/
Department of Plant Pathology Not satisfactory --------------
(Co-Advisor)
3. Dr. Shafaat Ahmad

Professor & Head Satisfactory/
(Member)
4. Dr. Sunil Zacharia

Assistant Professor Satisfactory/
(Member)
5. Dr.V. M. Prasad
Professor & Head Satisfactory/
Department of Horticulture Not satisfactory --------------
(Member)
The thesis has been examined by the Evaluation Committee and found acceptable.
Place: Allahabad
Date:
External Examiner Chairman
Evaluation Committee
SELF DECLARATION
This is to certify that I have personally worked on the research entitled

“Integrated Disease Management against Fusarium oxysporum f. sp. lycopersici on
Tomato (Lycopersicon esculentum L.)”. The data mentioned in the thesis were
recorded during the work and are genuine. None of the findings/information pertaining
to the work has been concealed. Any other data or information in this thesis, which has
been collected or borrowed from outside agency, has been duly acknowledged.
Date: HUSSEIN ALI SALIM
Place: ALLAHABAD ID. No. 12PHCPP103

Integrated Disease Management against Fusarium oxysporum
f.sp.lycopersici on Tomato (Lycopersicon esculentum L.)
Abstract
Integrated disease management (IDM) play a vital role in pest management and
hence have been widely used in agriculture. Several biocontrol agents including
Pseudomonas fluorescens and Trichoderma harzianum, Neem cake and Soil solarization
have been exploited in the management of soil-borne diseases. The infested soil of
Fusarium oxysporum f.sp. lycopersici of 6 microplots was light irrigated and covered
with an ultraviolet stabilized, 30 µm clear polyethylene film in 4 microplots, two
microplots was kept as nonsolarized soil (Fusarium infested). Four days before sowing
the seeds of tomato variety (CO-3), the solarized and unsolarized soil was mixed with
FYM @ 100 g / pot and filled in each pot 10 kg capacity of pot before application of
Trichoderma harzianum and Pseudomonas fluorescens @ 2 g / pot, carbendazim 50 %
W.P @ 2 kg a.i / ha, Neem cake powder @ 10 g/ pot and spent mushroom compost @
20 g / kg of soil. . Solarized soil with Neem cake and Carbendazim revealed that Neem
cake with Carbendazim significantly increased the plant growth parameters and
reduced disease intensity. The treatments of solarized soil with Pseudomonas
fluorescens and spent mushroom compost significantly increased the plant growth
parameters as compared with other treatments including control (F.o alone). The disease
intensity (%) were significantly reduced in T5 (Carbendazim + F.o), T4 (solarized soil +
Pseudomonas fluorescens + spent mushroom compost), T3 (solarized soil +
Pseudomonas fluorescens) and T2 (solarized soil + spent mushroom compost) as
compared with T1 (Solarized soil + F.o) and T0 (F.o alone) respectively. The treatments
of solarized soil with Trichoderma harzianum and spent mushroom compost
significantly increased the plant growth parameters as compared with other treatments
at 30, 60 ,90,120 and 150 days after germination. The disease intensity (%) were
significantly reduced in T5 (Carbendazim), T4 (solarized soil + Trichoderma harzianum
+ spent mushroom compost), T3 (solarized soil + Trichoderma harzianum) and T2
(solarized soil + spent mushroom compost) as compared with T1 (Solarized soil + F.o)
and T0 (F.o alone) respectively.
Key words: Tomato plant, Fusarium oxysporum f.sp. lycopersici, Carbendazim ,Neem
cake , Pseudomonas fluorescens , Trichoderma harzianum ,spent mushroom compost,
and Soil solarization.
Chapter - I
INTRODUCTION
Tomato (Lycopersicon esculentum Mill.) is an important vegetable crop belongs

to family solanaceae widely grown in the tropics. In tropical Asia, it is an important
cash crop for small farmers (Villareal, 1979).
The major tomato growing countries are China, USA, Italy, Turkey, India and
Egypt (Table. 1.1). Total area under tomato cultivation in India is about 3.56 lakh
hectare with an average yield of 15.7 tons per hectare (Raithak and Gachande, 2013).
Uttar Pradesh, Maharashtra, Karnataka, Bihar and Orissa are major tomato
growing states in India. The area under its cultivation is 865000 ha with a production of
16826000 tonnes, productivity 19.5 tonnes per ha contributing 7.95% of total area and
production. In vegetables production, India is next only to China with an annual
production of 87.53 million tonnes from 5.86 million hectares having a share of 14.4 per
cent to the world production. Vegetables form the most important component of a
balanced diet for human beings. Present production of 1.5 million tonnes of vegetable
supply only 145 g per capita per day against recommended requirements of 300 g.
(Shiva et al. 2013). There is a sizeable increase in acreage and production of tomato in
India while in terms of production it has increased from 10055.0 to 16826.0 thousand
tons (Table.1.2).
Tomato fruit is rich in source of minerals like iron and phosphorus, vitamins A
and C and organic acid, essential amino acids, dietary fibers and also contains lycopene
and Beta-carotene pigments, 3%~4% total sugar, 4%~7% total solids, 15~30 mg/100g
of ascorbic acid, 7.5~10 mg/100 mL titratable acidity and 20~50 mg/100g fruit weight
of lycopene .Tomatoes may be used as sauces, salads, ketchup, juices and side dishes
and it can be baked, roasted, boiled, or stewed. It plays an important role in maintaining
the human health. Being rich source of lycopene, tomato is used in the treatment of
cancer, especially the prostate cancer (Giovannucci, 1999; Shiva et al. 2013).
Tomatoes can make people healthier and decrease the risk of conditions such as
cancer, osteoporosis and cardiovascular disease. People who ate tomatoes regularly
have a reduced risk of cancer diseases such as lung, prostate, stomach, cervical, breast,
oral, colorectal, esophageal, pancreatic, and many other types of cancer. Lycopene and
NITCIDORTNIN 1
the newly discovered bioflavonoids in tomatoes are responsible as cancer fighting
agents. Not only raw tomatoes but also cooked or processed tomato products such as
ketchup, sauce, and paste, are counted as good source of cancer prevention. Tomato is
also good for liver health. Tomato has detoxification effect in the body. Probably it is
due to the presence of chlorine and sulfur in tomatoes. According to some studies, 51
mg of chlorine and 11 mg of sulfur in 100 grams size of tomato have a vital role in
detoxification process (Bhowmik et al, 2012).
Table. 1.1: Major Tomato producing Countries in the world
Source: Indian Horticulture Database, 2011
Table.1.2: The Area, Production and Productivity of Tomato in India
Year Area (000’ha) Production(000’tons) Productivity(tons/ha)
2006-2007 596.0 10055.0 16.9
2007-2008 566.0 10303.0 18.2
2008-2009 599.0 11149.0 18.6
2009-2010 634.4 12433.2 19.6
2010-2011 865.0 16826.0 19.5
Source: Indian Horticulture Database, 2011
The most common diseases of tomato include early blight, anthracnose, bacterial
wilt, bacterial canker, tomato spotted wilt, Verticillium wilt and Fusarium wilt , the wilt
diseases of tomato are caused by bacteria (Pseudomonas spp) and fungi (Fusarium and
Verticillium spp) (Mardi et al. 2002).
NITCIDORTNIN 2
Fusarial wilt caused by Fusarium oxysporum schlechtent.f.sp. lycopersici
(Sacc.) Synder and Hans is a destructive disease of tomato crops worldwide (Sheu and
Wang, 2006). This disease was first described by G.E. Massee 1895 in England (Mui-
Yun Wong, 2003). Shiva et al., (2013) stated that the disease was first reported in India
from Pusa Bihar as Fusarium oxysporum f.sp. lycopersici (Sacc).
F.oxysporum is a major problem in tomato crop in the warm, moist tropical
regions of the world (Aycock, 1966), causing damping-off in nursery seedlings as well
as stem rot, wilting and blight in adult plants (Tindall, 1983; Chamswarng et al. 1992;
Flores-Moctezuma et al. 2006), with consistent loss of production (De Curtis et al.
2010).
Fusarium oxysporum is a soil borne fungal pathogen that infects plants through
roots at all stages of plant growth, causes major economic losses by inducing necrosis
and wilting symptoms in many crop plants (Ozbay and Newman, 2004; El- Khallal,
2007).
The pathogen enters through the roots of the plant and proliferates in the vascular
tissues leading to breakdown of the water economy of the infected plants (Agrios,
2005). The disease causes great losses, especially on the susceptible varieties of tomato
when soil and air temperature are rather high during the warm season (Agrios, 2005;
Mandal et al. 2009).
Typical symptoms of the disease are yellowing and wilting of leaves progressing
upward from the base of the stem. Initially, only one side of a leaf mid rib, one branch,
or one side of a plant is affected. The symptoms soon spread to the rest of the plant and
finally kill it. Due to prolonged survival in soil as a saprophyte and as resistant
structures, F. oxysporum is difficult to control (Mujeebur and Shahana, 2002;
Borrero et al. 2004).
The loss in tomato yield by F. oxysporum varies between 10 to 90% depending on
the stage of the plant growth at which section occurs and the environmental conditions
(Singh, 2005; Kumar and Sood; 2002). Fusarium wilt is one of the most important
disease which highly destructive to tomatoes grown in greenhouse and in the field in
many warm regions of the world, where it causes 10-50 % yield loss (Larkin and
Fravel, 1998; Borrero et al., 2004). It is of worldwide importance where at least 32
countries had reported the disease, which is particularly severe in countries with warm
climate. Mui-Yun Wong, (2003). It is a devastating disease causing considerable
NITCIDORTNIN 3
economic losses ranging from 10-80% yield loss in many tomatoes producing area of
the country (Kesavan and Chaudhary, 1977). Increased early injury to the roots and
collar of tomato plants caused by Fusarium oxysporum was also observed in Tunisia,
where yield losses were reported to range between 20 and 60%, FORL causes 90% of
crop losses with repeated infections that result in microconidia spreading, especially in
the same growing season in greenhouses (Hajlaoui et al. 2001). There may be 60 to
70% yield loss due to this disease (Kumar Kiran et al., 2008). Fusarium wilt has been
the most devastating disease resulting in 10 to 50% crop losses around the world
(Lukyanenko, 1991). The yield loss due to this disease may be about 30 to 40%
(Kirankumar et al., 2008). In Manipur the vegetable growers suffer more than 50%
crop losses due to Fusarium wilt of tomato in heavily infested fields (Nirupama Devi et
al., 2013). Singh and Kamal, (2012) reported 10-90% loss in yield of tomato in
temperate region due to this disease.
Integrated disease management (IDM)

Integrated disease management (IDM), which combines biological, cultural,
physical and chemical control strategies in a holistic way rather than using a single
component strategy proved to be more effective and sustainable, the concept of
Integrated Disease Management (IDM) (El Khoury and Makkouk, 2010).
The IDM involves a total system approach to the suppression of pathogen
populations to a level where higher yields can be obtained and enables the farmers to
achieve maximum economic return. In the IDM system, the individual component of
disease management such as host plant resistance (HPR), agronomic practices, judicious
use of fungicides, pesticides for vector control, biopesticides for pathogen control etc.,
need to be compatible or complimentary (Pande et al., 2009).
Neem
Neem pesticides play a vital role in pest management and hence have been widely
used in agriculture. There has been an evident shift all over the world from synthetic
pesticides to non-synthetic ones; this is largely because of the wide spread awareness of
the side effects of these synthetic pesticides not only on plants and soil but also on other
living organisms. Neem pesticides are being manufactured and exported to various
countries as a lot of research has been conducted to test the safety and efficacy of neem
for use as a pesticide (Joseph et al., 2010; Vethanayagam and Rajendran, 2010).
NITCIDORTNIN 4
Azadirachtin is the main ingredient used to manufacture bio pesticides. Neem oil and
seed extracts are known to possess germicidal and anti-bacterial properties which are
useful to protect the plants from different kinds of pests. One of the most important
advantages of neem-based pesticides and neem insecticides is that they do not leave any
residue on the plant.
Neem and its products has been widely reported to control insect pests (Ascher,
1993), plant bacterial diseases (Abbasi et al. 2003), plant parasitic nematodes (Muller
and Gooch, 1982; Akhtar and Mahmood, 1995), plant fungal diseases (Vir and
Sharma, 1985; Amadioha, 2000; Dubey et al. 2009) and a potential agricultural
fertilizer (Gajalakshmi and Abbasi, 2004). Moreover, in ayurveda, unani and
homeopathic medicine almost every part of this tree including seeds, leaves, roots, bark,
trunk and branches has multiple uses (Subapriya and Nagini, 2005).
Srivastava et al.(1997) showed the fungicidal properties of aqueous leaf extracts
of Azadirachta indica against Alternaria alternata from pear fruits with 85 % control of
fruit rot in vivo. Patil et al. (2001) showed that neem leaf extract was effective in
reducing early blight incidence with increased yield of tomato infected by Alternaria
solani. Plant extracts have played significant role in the inhibition of seed-borne
pathogen F. oxysporum and in improvement of seed quality and emergence of plant
seeds (Nwachukwu and Umechuruba, 2001). Kishore et al. (2001) reported that
ethanol leaf extract of A. indica was highly inhibitory to Phaeoisariopsis personata, the
causal agent of late leaf spot of ground nut. Aboellil, (2007) reported that Trilogy, a
natural product from A. indica was significantly retarded several growth parameters of
cucumber powdery mildew pathogen (Podosphae raxanthii), and induced resistance in
cucumber plants.
Compost
The use of compost as a peat substitute to control root pathogens was first
suggested by several researchers (Hoitink et al. 1975; Scheverell and Mahaffee, 2004;
Litterick and Hamler, 2004; Segarra et al. 2009). The use of compost extracts to
control diseases in plants is an effective alternative that enables the use of pesticides to
be reduced (Haruna et al. 2012; Segarra et al. 2009).
Several soil-borne plant pathogens have been reduced by using composts made
of different raw materials (Borrero et al. 2004; Cotxarrera et al. 2002; Hoitink and
NITCIDORTNIN 5
Boehm, 1999; Hoitink and Fahy, 1986; Litterick and Hamler, 2004). The capacity
of certain composts to suppress Rhizoctonia solani may be due to the presence and
activity of specific antagonists (Kuter et al. 1983; Scheuerell et al. 2005; Tuitert et
al.1998) and depends on the degree of compost decomposition (Hoitink and Boehm,
1999). Moreover, matured compost can sustain biological control agents, whereas
immature compost does not support them, negatively affecting the growth of crop plants
and possibly containing pathogen populations (De Ceuster and Hoitink, 1999;
Litterick and Hamler, 2004).
The abiotic component of compost is an important factor for its suppressive effect
in soils. Suárez-Estrella et al. (2012) suggested that compost is considered a source of
biocontrol agents against several serious plant pathogens.
Suppression of soil-borne fungal diseases by means of organic amendments,
including compost, is reported by various scientists (Paulitz and Belanger, 2001;
Bailey and Lazarovits, 2003). The production of fungitoxic compounds such as
organic acids or ammonia from some organic amendments could enhance the
antagonistic mechanism involved in suppressing soil-borne fungi. These mechanisms
include competition for nutrients and/or growth of specific agents that evoke antibiosis
or induced resistance against plant pathogens (Hoitink and Boehm, 1999; De Clercq et
al. 2004).Several studies have shown that composts from heterogeneous sources can
suppress different plant diseases (Diab et al. 2003; Scheuerell et al. 2005; Suárez-
Estrella et al. 2007).
Raj and Kapoor, (1997) reported that mushroom compost have effect in
enhancing the microbial activity in the amended soil and the higher dosage (2%, w/w)
of composts are most effective in managing the pathogen Fusarium oxysporum f. sp.
Iycopersici, thereby resulting in better plant health and disease control.
Mushroom compost (spent mushroom substrate, SMS, mushroom soil) exhibits
suppressive characteristics against various fungi, as well as against plant diseases
caused by fungi. In addition, mushroom compost has physical and chemical
characteristics that make it ideal for blending with landscape mulch to enhance growth
of horticultural plants. (Davis et al. 2005).
Incorporation of composted Spent Mushroom Substrate (SMS) not only improves
the nutrient status but also neutralizes the acidity of soils (Pannier, 1993; Ahlawat et
al., 2005) and facilitates cultivation even in problem soils (Ahlawat et al., 2007). In
NITCIDORTNIN 6
addition, SMS also possesses good bio-control activity against certain foliar and soil
borne diseases (Yohalem et al., 1996; Ahlawat et al., 2007b) and potential to
bioremediate several agricultural grade 88 fungicides and pesticides (Ahlawat et al.,
2010; Ahlawat et al., 2011). SMS, after suitable pretreatments can completely or
partially substitute the growing media for cultivation of horticultural crops of economic
importance (Beyer, 1996; Kaddous and Morgans, 1986).
Bio-agents
Biological control agents (BCAs) are currently being examined as alternatives to
the synthetic pesticides due to their perceived increased level of safety and minimal
environmental impacts (Cotxarrera et al. 2002; Brimner and Boland, 2003).
Several biocontrol agents including Pseudomonas fluorescens, Pseudomonas
putida, Trichoderma harzianum and Bacillus subtilis have been exploited in the
management of soil-borne diseases (Ongena et al. 1999; Yigit and Murat, 2007;
Jayaraj et al. 2007). The bacteria effectively reduced Fusarium wilt incidence on
tomato under glasshouse conditions. Several bacterial and fungal antagonistic strains
have been shown to reduce the damage caused by different pathogens, including F.
oxysporum f. sp. lycopersici (Postma and Rattink, 1992; Alabouvette, 2001; Moretti
et al. 2008).
Trichoderma harzianum
The genus, Trichoderma is common filamentous imperfect fungi (Deutromycetes,
Dematiaceae), the most common saprophytic fungi in the rhizosphere and found in
almost any soil. The mycoparasite ability of Trichoderma species against some
economically important aerial and soil borne plant pathogens (Papavizas, 1985; Elad et
al. 1993; Elad, 2000; Freeman et al. 2004, Dubey et al. 2007) and nematodes
(Windham et al. 1989; Sharon et al. 2001) allows for the development of biocontrol
strategies. Several Trichoderma species reduces the incidence of soil borne plant
pathogenic fungi under natural conditions (Sivan and Chet, 1986; Calvet et al. 1990);
however the efficacy of this depends largely on the physical, chemical and biological
condition of soil. There have been numerous recent attempts to use Trichoderma spp on
soil borne pathogens such as Sclerotinia, Fusarium, Pythium, and Rhizoctonia species
in world (Elad et al. 1980; Jager et al. 1991; Ashrafizadeh et al. 2005; Dubey et al.
2007). Among these, several species of Trichoderma are well documented mycoparasite
NITCIDORTNIN 7
and have been used successfully against certain pathogenic fungi (Papavizas and
Lumsden , 1980). T. harizanum, T. viridae, T. virens, T. hamatum, T. roseum and T.
koningii are the species that most often used biological control of pathogens.
Harman, (2006) developed Trichoderma spp into several commercial biological
control products and is used in field crop and greenhouse system. These products are
known to control numerous soil-borne diseases such as Fusarium oxysporum, Pythium
and Scelerotinia, and also enhance plant growth (Neseby et al. 2002; Rojo et al. 2007),
was also suggested that, Trichoderma affects induced systemic resistance mechanism in
plants against pathogens (Haggag, and Amin, 2001; Prasad et al. 2002; Abd-El-
Kareem, 2007; Hibar et al. 2007; Rojo et al. 2007; Jayalakshmi et al. 2009).
Pseudomonas fluorescens
Pseudomonas spp. are common soil bacteria easily cultured from most
agricultural soils and plant rhizospheres. They have been studied in considerable detail
because of their ability to promote plant growth, either by directly stimulating the plant
or by suppressing pathogens (Haas and Defago, 2005; Carlier et al. 2008; Rovera et
al. 2008; Rosas et al. 2009; Ross et al. 2000; Srinivasan et al. 2009).Several research
groups have tested the efficacy of antagonistic microbes for the control of F.oxysporum
(Madi et al. 1997; Tsahouridou and Thanassoulopoulos, 2002; Errakhi et al.
2007).which induces diseases difficult to control because of the production of sclerotia
that represent resistant survival structures (Elad, 1995). Fluorescent pseudomonads
have been reported as promising biological control agents against F. oxysporum in betel
vine (Singh et al. 2003).
Elad and Chet, (1987) reported several isolates of P. fluorescens, P.putida, P.
aeruginosa and P. aureofaciens suppress the soil-borne pathogens through rhizosphere
colonization antibiosis (Pierson and Thomashow, 1992) and iron chelation by
siderophore production (Lemanceau et al. 1992). Certain fluorescent pseudomonads
are also found to promote plant growth by production of plant growth-promoting
substances and thus they are called plant growth promoting rhizobacteria (PGPR).
PGPR are known to induce resistance against fungal, bacterial, viral diseases and insect
pests (Chen et al. 2000; Maurhofer et al. 1998; Wei et al. 1996; Zehnder et al. 1997).
NITCIDORTNIN 8
Soil solarization
Soil solarization is a natural hydrothermal process of disinfesting soil of plant

pests and pathogens that is accomplished through passive solar heating. Solarization is
commercially practiced mainly in areas which are characterized by high summer air
temperatures such as the Mediterranean, deserts, and tropical areas and is affected by
several factors, including solar irradiation intensity, air temperature, plastic color and
type, soil moisture, soil properties and other factors (De Vay and
Katan, 1991; Stapleton, 2000). The most well-known function of solarization is
reduction of soilborne inoculum of plant pathogens including fungi, bacteria, and
nematodes by direct thermal inactivation which is achieved at soil temperatures ranging
from 40°C to more than 60°C (Gelsomino et al., 2006). In addition, soil solarization
increases the release of soluble nutrients (inorganic N forms, extractable P, and K,
available cations, and dissolved organic matter) due to soil heating, and consequently
results in improved plant growth and yield increases (Gelsomino et al., 2006; Stevens,
et al. 1991).
Many efforts are being made to improve solarization efficiency in controlling
soilborne pathogens including integration with a biological control agent, lower dosages
of chemical fungicides, organic amendments (composts, plant residues, and green and
animal manures), and physical methods (plastic mulch type, and double-layer mulch)
(Stapleton, 2000; Stevens et al., 2003). Using a double layer of polyethylene sheets
makes soil solarization more feasible in areas with cooler climates and increases soil
temperatures 2–5°C more than a single layer (De Vay and Katan, 1991).Several
management options have been suggested to control Tomato wilt disease, including soil
solarization (Ioannou et al., 2000;Tamietti and Valentino, 2006).
Carbendazim
Carbendazim, one of the fungicide was approved by International Standardization

Organization (ISO) with a common name for methyl 2-benzimidazole carbamate, a
systemically active benzimidazole fungicide that inhibits the synthesis of ᵦ-tubulin
(Pfeil and Dellarco, 2005). The benzimidazole fungicide carbendazim was used for the
control of many diseases caused by Deuteromycetous pathogens (Delp and Klopping,
1968), owing to its systemic properties and its great efficacy in controlling plant
diseases. It was also recommended for a long period for the control of ripe rot disease of
NITCIDORTNIN 9
chillies (Capsicam annum L.) caused by C. capsici (Syd.) (Bollen and Zaayen Van,
1975; Delp, 1980; Georgopoulos and Dovas, 1973; Miller and Fletcher, 1974).
However, although some existing studies conducted on inhibitory effects of
carbendazim fungicide on mycelial growth of either Fusarium or Colletotrichum, in the
current literature there are only few or no reports about the influence of Carbendazim
fungicide on mycelial growth of F.oxysporum f.sp. lycopersici from Tomato plant and
C. capsici from Chilli plant.
Amini and Dzhalilov,(2010) reported that six fungicides; benomyl, carbendazim,
prochloraz, fludioxonil, bromuconazole and azoxystrobin,were evaluated for their
efficacy against the disease causal agent Fusarium oxysporum f. sp. lycopersici in vitro
and in vivo. Seven different concentration (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 μg/ml)
were used for assessment of their inhibitory activities against the pathogen through
mycelial growth inhibition on potato media. Four concentrations of above mentioned
fungicides (0.1, 1. 10 and 100 μg/ml) were tested for controlling Fusarium wilt on
tomato plants in glasshouse. Fungal radial growth was measured and median effective
concentration (EC50) values (μg/ml) determined.
In view of the above the present investigation “ Integrated Disease Management

against Fusarium oxysporum f.sp. lycopersici on Tomato (Lycopersicon esculentum
L.)’’ was undertaken with the following objectives-
1- To carry out pathogenicity test of Fusarium oxysporum f.sp. lycopersici by

following Koch’s postulate.
2- To evaluate the effect of solarized soil with Neem cake and Carbendazim on
disease intensity of wilt incidence causes by Fusarium oxysporum f.sp.
lycopersici and plant growth parameters and yield of tomato.
3- To evaluate the effect of carbendazim and solarized soil with Pseudomonas
fluorescens, Trichoderma harzianum, spent mushroom compost on Fusarium
oxysporum f.sp. lycopersici and plant growth parameters and yield of tomato.
NITCIDORTNIN 10
Chapter - II
REVIEW OF LITERATURE
Fusarium oxysporum f. sp. lycopersici
Reis et al. (2005) reported that three races of Fusarium oxysporum f. sp. lycopersici, are
the most important races of tomato (Lycopersicon esculentum). Races 1 and 2 are
distributed worldwide whereas race 3 has a more limited geographic distribution with
no report thus far in Brazil. Virulence assays were performed using a set of the race
differential cultivars: ‘Ponderosa’ (susceptible to all races), ‘IPA-5’ (resistant to race 1),
‘Floradade’ (resistant to races 1 and 2) and ‘BHRS-2, 3’ (resistant to race 3). All
isolates were highly virulent to ‘Ponderosa’, ‘IPA-5’ and ‘Floradade’ and were able to
infect only a few plants of ‘BHRS- 2, 3’. An additional virulence test was conducted
including the same set of cultivars plus Lycopersicon pennellii ‘LA 716’. Identical
results were obtained with L. pennellii displaying an extreme (immune-like) resistant
response.
Shishido et al. (2005) examined the efficiency of nonpathogenic Fusarium oxysporum

Fo-B2 for the biological control of Fusarium wilt of tomato, caused by F. oxysporum f.
sp. lycopersici CU1, in different environments: a growth chamber with sterile soil-less
medium, a greenhouse with fumigated or non-fumigated soil, and non-fumigated field
plots. Inoculation of Fo-B2 onto tomato roots significantly reduced the severity of
disease, but the efficiency of disease suppression decreased as the experimental
environment became less controlled. Relationships between the recovery of Fo- B2
from hypocotyls and the disease severity indicated that the biocontrol agent was most
effective when it colonized vascular tissues intensively. Moreover, the degree of Fo-B2
colonization was greatly reduced when the seedlings were grown in non-fumigated soil.
Anitha and Rabeeth (2009) reported that the production of tomato and pepper fruit is
of worldwide agricultural importance. Many diseases and disorders can affect tomatoes
during the growing season. Fusarium oxysporum f. sp. lycopersici (FOL) is a highly
destructive pathogen of both greenhouse and field grown tomatoes in warm vegetable
ERUFRR TI FIREEIVER 11
production areas. The disease caused by this fungus is characterized by wilted plants,
yellowed leaves and minimal or absent crop yield. There may be a 30 to 40% yield
losses due to this disease.
Srinon et al. (2006) isolated Fusarium oxysporum f.sp. cucumerinum and F. oxysporum
f.sp. lycopersici (plant pathogens) from cucumber and tomato and those virulence
isolate’s activities were inhibited using several antagonisitic fungi by bi-culture test.
Most of antagonistic fungi effectively inhibited the spore production of the plant
pathogens where the growth inhibition of the spore production was greatly increased in
some of the antagonistic fungi used. They found that Trichoderma hamatum WS01 and
Penicillium sp.WS01 showed a higher diameter of clear zone than the other fungi on
cellulose activity.
Chellemi et al. (2012) studied five diverse land management practices were for their
impacts on Fusarium wilt (Fusarium oxysporum f. sp. lycopersici), galling of roots by
Meloidogyne spp. and marketable yield of tomato (Solanum lycopersicum) and to
identify associations between the severity of pest damage and the corresponding soil
microbial community structure. The incidence of Fusarium wilt was >14% when tomato
was cultivated following 3 to 4 years of an undisturbed weed fallow or continuous
tillage disk fallow rotation and was >4% after 3 to 4 years of bahiagrass (Paspalum
notatum) rotation or organic production practices that included soil amendments and
cover crops. The incidence of Fusarium wilt under conventional tomato production with
soil fumigation varied from 2% in 2003 to 15% in 2004. Increased Fusarium wilt by
20% or more except when tomato was grown using organic practices, where disease
remained less than 3%. The percent of tomato roots with galls from Meloidogyne spp.
ranged from 18 to 82% in soil previously subjected to a weed fallow rotation and 7 to
15% in soil managed previously as a bahiagrass pasture. Repeated tomato cultivation
increased the severity of root galling in plots previously subjected to a conventional or
disk fallow rotation but not in plots managed using organic practices, where the
percentage of tomato roots with galls remained below 1%. Marketable yield of tomato
exceeded 35 Mg ha–1 following all land management strategies except the strip-
tillage/bahiagrass program. Marketable yield declined by 11, 14, and 19% when tomato
was grown in consecutive years following a bahiagrass, weed fallow, and disk rotation.
The composition of fungal internal transcribed spacer 1 (ITS1) and bacterial 16S rDNA
amplicons isolated from soil fungal and bacterial communities corresponded with
observed differences in the incidence of Fusarium wilt and severity of root galling from
Meloidogyne spp. and provided evidence of an association between the effect of land
management practices on soil microbial community structure, severity of root galling
from Meloidogyne spp., and the incidence of Fusarium wilt.
Ignjatov et al. (2012) performed the isolation and morphological determination of the
fungus on PDA and Czapek's nutrient media. Isolates from diseased plants growing in
field, designated as TFW1-TFW12 and seven isolates from diseased tomato fruits
(TFM1-TFM7) were chosen for further investigation. For identification of the fungal
isolates, the polymerase chain reaction (PCR) was also used. The EF1/EF2 primer pair
was used for molecular identification of Fusarium sp. nine analyzed samples were
found to contain DNA fragments 700 by in size.
Nirmaladevi1 and Srinivas (2012) isolated Fusarium oxysporum f. sp. lycopersici

strains from wilted tomato plants and soil samples collected from different tomato fields
in and around Karnataka. A total of 114 isolates named as (MB1-MB114) were
subjected to cultural, morphological and pathogenicity studies. Significant variations
existed among the isolates with respect to rate and type of growth, colony colour,
mycelial growth pattern, sporulation, septation of the conidia, number and pattern of
chlamydospore formation and their pathogenic variability by inoculating to five
different tomato varieties by standard root dip method. The pathogenicity of the isolates
could be categorized based on their virulence. The invasive growth of the isolates was
assayed by injecting the conidial suspension of the isolates to tomato fruits.
Vethavalli and Sudha (2012) assessed the disease incidence caused by Fusarium
oxysporum f.sp. lycopersici. The maximum incidence of 30 percent was recorded in
Thondamuthur village of Coimbatore District followed by Ayyalur village of Dindigul
District. Among the thirty isolates isolated from rhizosphere soil collected from two
Districts, Pseudomonas sp. (PV2) recorded the maximum inhibition zone of 16 mm and
66.16 per cent inhibition; Bacillus sp. (BVE1) recorded 18mm of inhibition zone, 68.33
percent over control and Serratia sp. (SM1) recorded the maximum inhibition zone of
15.20 mm and 64.44 per cent inhibition of mycelia growth over control in vitro. The
highest inhibition 88.33 per cent over control was observed in BVE1 isolates revealed
that the isolate inhibited the growth of Fusarium oxysporum f .sp. lycopersici. The
efficiency of Bacillus antifungal compound proteins have an inhibitory activity towards
receptor fungal protein of Polygalacturonase.
Raithak and Gachande (2013) isolated pathogenic fungi, Fusarium oxysporum f.sp.
lycopersici and Alternaria solani (Elis and Mart) Sorauer from infected tomato roots
and fruits respectively from different varieties of tomato. The effect of culture filtrate of
these fungi was observed on seed germination, root, and shoot length and vigour index.
Culture filtrate obtained from F. oxysporum f. sp. lycopersici isolated from tomato
varieties Laxmi (NP-5005) increased seed germination up to 80 % vigour index 904.0,
root length 6.14 cm and shoot length 5.16 cm as compared to other varieties. A. solani
isolated from var. Priya (BSS-908) increased seed germination up to 70%, vigour Index
784.7, root length 5.93 cm and shoot length 5.28 cm as compared to other varieties.
Biological control
Gnanamanickam et al. (2002) reported that successful biological control systems

commonly employ and naturally occurring, antagonistic microorganisms that are able to
reduce the activities of plant pathogens. Such antagonists can compete with pathogens
for nutrients, inhibit pathogen growth by secreting antibiotics, or reduce pathogen
populations through parasitism. In addition, some of these microorganisms induce
resistance in host plants, which enhances the plant’s ability to defend itself from
pathogen attack.
Shiva et al. (2013) studied biological control of Fusarium oxysporum f.sp. lycopersici
and Pythium aphanidermatum causing wilt and damping off in order to evaluate the
relative water content, soluble protein content, total phenolic content and yield changes.
Application of P.fluorescens + T.viride + soil application had higher soluble protein
content and total phenolic content in tomato. The highest increase in soluble protein and
total phenolic content of 20 and 18 per cent over control leads to very lesser reduction
in yield by the application of P.fluorescens + T.viride + soil application during the
presence of wilt and damping off diseases.
Neem
Kimaru et al. (2004) investigated effect of Neem Kernel Cake Powder (NKCP) at 1.75,
3.5 and 7g rates on development of tomato Fusarium wilt. Performance of plants grown
in NKCP amended and non-amended soils was significantly (p=0.05) different (33.3 -
93.3%). Disease severity based on the wilt index (0.53-2.87) and length of discolored
vascular tissues (7.4cm - 25.62cm) differed significantly (p=0.05) among treatments.
Mukhtar (2007) evaluated the effect of four plant aqueous extracts species viz;
Azadirachta indica A. Juss., Datura metel. var. quinquecuspida Torr., Ocimum sanctum
L. and Parthenium hysterophorus against F. oxysporum f. sp. ciceri . It was found that
all the plant extracts at 40% concentration were effective in reducing the mycelial
growth of F. oxysporum f. sp. ciceri. Among these plants extracts, A. indica and D.
metel inhibited fungal growth up to 80% at 10% concentration. Chemical treatment with
Benomyl (50WP) and Carbendazim (50WP) was proved to be the most effective against
F. oxysporium f. sp. ciceri.
Hassanein et al. (2008) evaluated leaf extracts of neem (Azadirachta indica) and
chinaberry (Melia azedarach) against Alternaria solani and Fusarium oxysporum, the
inhibition percentages of the neem were 17.88%, 23.66, 52.77 % and 70.55% for
Alternaria solani in the four used concentrations, while those for F. oxysporum were
14.77 %, 23.88%, 31.22 % and 100%, respectively. The corresponding values with
chinaberry leave extracts were 3.11 %, 5.22%, 5.53 % and 5.77 %, recorded for Alt.
solani and 5.44 %, 6.11 %, 6.35 % and 6.55% for F. oxysporum. The concentrations of
neem extract effectively suppressed the mycelial growth of both pathogenic fungi and
this effect gradually increased with increasing concentration. In addition, tests on
activity of the main component, nimonol purified by HPLC as a separate component
from neem ethyl acetate extract (mother extract), it gave no inhibition activities at all
concentrations. Pathogenicity tests of Alternaria solani showed disease incidence of
53.20 and 100 % after 2 and 4 weeks. Spraying tomato plants with 20% aqueous neem
leaf extracts lowered the disease incidence to 42.54%.
Farag Hanaa et al. (2011) investigated the effect of neem (Azadirachta indica) and
willow (Salix babylonica) aqueous extracts on Fusarium wilt disease in tomato
seedlings. Four weeks old tomato seedlings were treated with 10% of either neem and
willow aqueous extracts and then inoculated with Fusarium oxysporum after 4 days of
treatment. The results showed that the percentage of disease incidence was increased in
non-treated tomato seedlings and reached the maximum level (65%) after 6 weeks of
infection. Treatment of tomato plants with neem and willow aqueous extracts reduced
the percentage of disease incidence to the level of 25.5% and 27.8% respectively.
Moreover, application of neem and willow aqueous extracts with Fusarium,
significantly reduced the level of lipid peroxidation and induces high activities of
antioxidant defensive enzymes after 3 and 7 days of infection. Electrophoretic pattern of
POX demonstrated that Fusarium oxysporum caused up regulation of several POX
isoenzymes. It could be concluded that neem and willow aqueous extracts reduced the
disease incidence of Fusarium wilt in tomato seedlings by increasing the activities of
antioxidant defensive enzymes and decreasing the level of lipid peroxidation.
Haruna et al. (2011) tested three different concentrations of three compost extracts-
poultry manure based compost extracts (PME), cow dung based compost extracts
(CDE), and neem-leaf based compost extracts (NLE). In vitro test was carried out to
evaluate the efficacy of the compost extracts at different concentrations on growth of
the pathogen. All the tested compost extracts significantly inhibited the mycelial growth
of the pathogen. Poultry manure compost extract and CDE applied as 1kg compost in
5L of distilled water induced the least radial growth (2.4cm) and highest percent
inhibition (46.5%) than the other compost extracts.
Rajput et al. (2011) tested the efficacy of different neem (Azadirachta indica A. Juss)
products namely neem oil, neem seed decoction, neem seed without coat, neem seed
coat and neem leaf extract in vitro growth of shisham seedlings inoculated with
Fusarium solani. Neem products used as spray increased the growth of inoculated
shisham seedlings as compared to injected at root zone or mixed with soil. Neem oil
(15%) used as spray increased root and shoot length and weight of inoculated shisham
seedling followed by neem seed decoction, neem seed without coat and neem leaf
extract as compared to untreated and inoculated shisham plants.
Hadian et al. (2011) observed Tomato inoculated with Meloidogyne and Fusarium with
50 g/kg soil neem seed powder significantly (P # 0.01) reduced the disease severity of
Fusarium and root-knot nematode. All the treatments significantly improved the growth
of the plants as compared to untreated inoculated plants. Carbofuran was highly
effective against nematode, Bavistin against fungus, Azadirachta indica seed powder
against both the pathogens. Neem decrease root knot index from 4.7 in control treatment
(fungi+nematode) to 0.25, and also decrease disease severity from 85% in control
treatment to 12%. Neem not only controlled these diseases but also cause increase in
growth characters such as plant weight and length.
Obongoya et al. (2009) evaluated fungitoxic properties of four locally available crude
plant extracts in controlling Fusarium oxysporum Schl. f.sp. phaseoli,. Crude plant
extracts from Azadirachta indica, Tagetes minuta, Nicotiana tobacum and Vinca rosea
were tested against Fusarium oxysporum Schl. f.sp. phaseoli. Minimum Inhibitory
Concentration (MIC) of crude plant extracts against Fusarium was determined by broth
micro dilution method. Crude plant extracts exhibited fungitoxic activity with varying
degree of efficacy, whereas Nicotiana tobacum and Vinca rosea were not effective,
Azadirachta indica and Tagetes minuta exhibited significant control over Fusarium.
Azadirachta indica performed better amongst all the plant extracts. Common bean
treatment with Benomyl 1 significantly reduced (P≤0.05) wilt incidence and increased
growth in comparison to negative (–ve) control. Azadirachta indica formulation gave a
significant reduction in wilt incidence compared to the other three crude plant extracts
formulations.
Haruna et al. (2012) evaluated the effect of compost extracts in suppressing Fusarium
wilt on three tomato varieties. The treatments consisted poultry manure-based compost
extract (PMCE), neem leaf-based compost extract (NLCE), cow dung-based compost
extract (CDCE), a synthetic fungicide (TEAM®) and sterile distilled water serving as
the control; and three tomato varieties (Roma VF, Duck Sekarat and UC 82B). Disease
incidence and severity was significantly (P≤0.05) lower on tomato plants treated with
the respective compost extract than on untreated plants. TEAM® was only effective at
the early stages of infection compared to the various extracts which were effective
starting from 6 – 7 weeks after transplanting till harvesting. However, poultry and cow
dung based- compost extracts were the most effective in reducing incidence and
severity of the disease. Higher yields were obtained with the application of PMCE (3.2 t
ha-1) and CDCE (3.0 t ha-1) in comparism with other treatments. It is therefore
recommended that farmers should use Roma VF and soil drenching with PMCE and/or
CDCE in an integrated pest management package in the control of Fusarium wilt of
tomato.
Arzoo et al. (2012) assessed the potentiality of different plant extracts like bark of
Eucalyptus Ianceolotus and Terminalia arjuna, tubers of Cyperus rotundus, leaves of
Withania somnifera, Azadirachta indica ,Datura stramoniurn, Acacia arabica.
Cymbopogon flexuosus and Parthenium hysterophorus. cloves of Allium sativun, bulb
of Allium cepa, fruits of Emblica officinalis and rhizome of Zingiber officinaIe as
inducers were assessed on physiological and biochemical activities in tomato against
Fusarium mysponma f sp. Iycopersici and the results showed that pre-application of
inducers provided protection to the tomato plant and reduced the disease intensity. The
minimum disease intensity (8.93%) was reported from garlic extract treated plant
whereas. in case of control-I it was 96.12% Treatment with plant extracts as induces
prior to challenge inoculation sensitized the seedlings to produce increased levels of
soluble protein The maximum increase in protein content was found in garlic extract
treated seedlings (32.62 mg g-l) after 15 days of pathogen inoculation. A high content of
phenols which are indicators of first stage of defence mechanism was also recorded in
treated leaves with maximum in garlic extract treatment representing 2. 28 mg g-l of
fresh leaves against 1.52 mg g-l of fresh leaves in control-II after 15 clays of pathogen
inoculation. The soluble protein content (r = -0.5995) and total phenol content (r = -
0.5313) both showed a negative correlation with disease incidence, Apart from inducing
effect in plant defences. Plant extracts have also some direct effect on growth and
development of the pathogen. Protein profiling by SDS-PAGE revealed that one new
protein is synthesized due to effect of induces that might be responsible for disease.
Compost
Raj and Kapoor (1997) used different composts viz., banana leaves, bagasse, synthetic
mushroom compost, paddy straw and spent mushroom compost at 0.5, 1.0 and 2% of
soil (w/w) as soil amendments against tomato wilt pathogen (Fusarium oxysporum f. sp.
Iycopersici (Sacc.) Suyder and Hansen), soil microflora, disease incidence and plant
growth in transplantend tomato plants in pots (cv. 'Pusa Ruby'). The various composts
used in the present investigation (6 weeks after transplant) reduced disease index by 44-
74%, brought down disease incidence from 89% in the control plants to 4-72% in
treated plants and mortality by 75-100%. The composts, in general, enhanced microbial
activity (total fungi and bacteria) in amended soils resulting in reduction in inoculums
density and capacity as well as better plant growth (in terms of shoot and root lengths)
and disease control. Banana leaves, mushroom and bagasse composts (2%, w/w) were
most effective in reducing the mean inoculum load by more than 78 (78.2 to 80.5) per
cent and increasing total fungal and bacterial population in the soil by 56.6 and 69.2
(mean 62.2), and 42.7 and 61 (mean 48.8) per cent, respectively, indicating enhanced
microbial activity. These composts (at 2% concentration) reduced disease index by 67
to 74%, disease incidence by 44-96% (maximum in banana leaves compost) and
prevented mortality completely. Composts, in general, exhibited stronger protection at
higher than at lower concentrations.
Borrero et al. (2005) evaluated olive oil husk + cotton gin trash composted and mixed
with rice husk (OC+R), spent mushroom composted and mixed with peat (SM+P) and
coir fiber. To determine the role of the microflora in the suppression, the two composts
were also heated to 60ºC for 6 days. Three bioassays were carried out with these
infested growth media and controls not infested. Disease severity was recorded during
25 days after tomato seedling transplantation.The two composts showed
suppressiveness to Fusarium wilt with respect to vermiculite and peat while coir fiber
was conductive. Furthermore, in OC+R its suppressiveness is due more to the growth
media’s non-heat-labile properties than in SM+P. Significant negative correlations were
found between severity and Bacillus spp., cellulolytic actinomycetes and bacteria,
oligotrophic actinomycetes and bacteria, copiotrophic bacteria, total actinomicetes
(cellulolytic + oligotrophic + copiotrophic) and total bacteria (cellulolytic + oligotrophic
+ copiotrophic) populations in these growth media. Bacillus spp. are known antagonists,
so they may account for the suppressiveness in our composts. Cellulolytic and
oligotrophic actinomycete populations were associated with Fusarium wilt
suppressiveness for another growth media. High microbial activity has been reported as
a Fusarium wilt suppressiveness factor. Copiotrophic, celullolitic and oligotrophic
bacteria contribute to this microbial.
Trillas et al. (2006) tested composts aged 0.5–1 year, cork compost reduced diseases
caused by Rhizoctonia solani in cucumber plants (53% of diseased plants) in
comparison to peat (up to 89%). However, all composts aged 1.5–3 years (comprised of
cork, grape marc, olive marc and spent mushroom) highly suppressed the disease
caused by Rhizoctonia sp. Plant growth media enriched with the biological control agent
Trichoderma asperellum (strain T-34) reduced the incidence of R. solani disease when
amended @ 103 c f u ml¡1. In composts aged 0.5–1 year, T-34 was only efficient when
added to spent mushroom and cork compost, although it remained well established in all
of them. The fact that T-34 rendered all composts aged 1.5–3 years highly suppressive
is attributed to the low levels of easily biodegradable substances. Rhizoctonia damping-
off in cucumber plants can be reduced by using composts and/or the biological control
agent T. asperellum strain T-34. In addition, the extent to which composts suppress this
disease depends on the nature of the composted materials, increasing with the composts’
maturity level.
Borrero et al. (2009) studied the suppressiveness of seven plant growth media to
Fusarium oxysporum f. sp. dianthi was in bioassays with carnation (Dianthus
cariophyllus) cv. Medea.These media were: grape marc compost, cork compost, olive
oil husk + cotton gin trash composted and mixed with rice husk, spent mushroom
compost mixed with peat, coir fiber, light peat and vermiculite. The compost media
showed a range of suppressiveness in comparison with peat.Grape marc compost was
the most effective plant growth medium in suppressing carnation Fusarium wilt. On the
other hand coir fibre, peat and vermiculite were conducive for this disease. β-
glucosidase activity and pH were positively correlated with disease severity as in other
reports for tomato.
Ahlawat et al. (2011) assessed the influence of button mushroom spent substrate (SMS)
on yield, quality and disease incidence by Fusarium wilt of pea (Pisum sativum). The
influence of SMS was compared with FYM and recommended inorganic fertilization
along with control. The 18 months old roadside discarded SMS supported highest
vegetative growth of pea during 2 months of initial experimentation. SMS decomposed
by aerobic and anaerobic methods for 9 to 18 months supported vegetative growth
almost at par with recommended dose of inorganic fertilization. The economic pod yield
was significantly higher (193.85% higher from control) in 18 month old an aerobically
composted SMS over the FYM, recommended inorganic fertilization and control.
Quality attributes like Ascorbic acid, Total soluble solids (TSS) and protein content
were either higher or almost at par with recommended fertilization. Incidence of
Fusarium wilts and powdery mildew was recorded lowest in 18 months old
anaerobically composted SMS treatment.
Estrella et al. (2012) used some kinds of compost suppress soil-borne plant pathogens
and although their exact mode of action is not yet clear, several mechanisms seem to be
involved in the suppressive effect. Results have shown that the compost’s suppressive
effect on mycelial development of Fusarium oxysporum f. sp. melonis (FOM), of
melon, was due to a combination of biotic and abiotic factors. Compost extracts were
used against the pathogenic fungus. In this case, compost extracts with a concentration
between 0.1 and 50% inhibited FOM growth. However, sterilized compost extracts
inhibited fungal development when applied at a dose lower than 10%, while higher
concentrations (>10%) were not effective, probably due to the toxic substances and
nutrient content ratio in the culture media.
Trichoderma harzianum
Montealegre et al. (2005) evaluated Trichoderma harzianum 650 (Th650) and

Paenebacillus lentimorbus 629 (Pl629) against root damage caused by F. oxysporum
f.sp. lycopersici - Pyrenochaeta lycopersici and also for R. solani in the summer assay.
methyl bromide (MeBr) decreased tomato root damage caused by the complex from
88.7% to 21.2% and from 78.4% to 35.7% in the summer and in the winter assay,
respectively. None of the biocontrollers could replace MeBr in the winter assay, but
Trichoderma harzianum 650 Th650 and Paenebacillus lentimorbus 629 Pl629 reduced
root damage caused by this complex in the summer assay. Treatments with
biocontrollers were improved by their combination with solarization in this season.
Independent evaluations showed that the positive control of Th650 towards R. solani
and the lack of effect on P. lycopersici correlates well with the endochitinase pattern
expressed by Th650 in response to these phytopathogens. Root damage caused by R.
solani can be controlled at a similar level as it does MeBr in summer assays, thus
representing an alternative to the use of this chemical fungicide for the control of this
phytopathogen.
Hibar et al. (2007) carried out an experiment and reported that Trichoderma spp.
significantly reduced disease incidence of Fusarium oxysporum f.sp. lycopersici
especially when they were applied one week before inoculation with the pathogen.
Light photomicrograph of samples from tomato roots treated with Trichoderma spp.
showed elaboration of structural barriers in regions situated within striking distance of
the pathogen penetration, formation of cell wall thickenings and occlusion of
intercellular spaces by a densely stained material, preventing thus pathogen evolution
and penetration.
Hajieghrari et al. (2008) evaluated six selected Iranian isolates of three species of
(Trichoderma hamatum T614, T. hamatum T612, T. harzianum T447, T. harzianum
T969, Trichoderma virens T523 and Trichoderma sp.) against five isolates of soil borne
phytopathogenic fungi (Fusarium graminearum, Rhizoctonia solani (AG4 and AG5),
Macrophomina phaseoli and Phytophtora cacturum) in dual culture techniques and
through production of volatile and non-volatile inhibitors, all Trichoderma isolates had
a marked statistical inhibitory effect on mycelial growth of the pathogens in dual culture
compared with controls. Maximum inhibitions occurred in F. graminearum-T. hamatum
T614 interaction. Significant pathogen colony growth inhibitions were observed when
exposed to the trapped atmosphere from culture of the Trichoderma. F. graminearum
was most susceptible to the volatile inhibitors produced by T. hamatum T612
(%inhibition = 48.65). Medium filtrate obtained the Trichoderma isolate culture also
were effected on the pathogen species significantly. Maximum growth inhibition was
observed in radial growth of F. graminearum by T. hamatum T612 nonvolatile
metabolites (%inhibition = 38.3). Evaluation of pH and temperature effects on
Trichoderma isolates mycelial growth showed that Trichoderma strains were found to
be able to display activities under a wider range of pH values. Also, Trichoderma strains
are mesophilic.
Morsy et al. (2009) reported that Trichoderma and Bacillus genera are most feasible
bio control microorganisms which suppress several pathogens like Fusarium solani.
The efficiency of these antagonistic' treated plant by strains was evaluated using an in
vitro assay. In pot experiment, the T. viride and/or B. subtilis suppressed F. solani as
indexed by survival rate. Field experiment was carried out at El-Fayoum Farm Research
Station during 2007 and 2008 seasons. Their results showed that, these treatments
favoured greater proliferation of rhizosphere microflora and higher dehydrogenase
activity in the rhizosphere. The dual treatment by T. viride + B. subtilis decreased the
percentage of infection and increased survival rate than individual one. Moreover, the
dual inoculation gave the highest records of growth parameters, fruit yields and plant
nutrient content than individual one. Thus, it is recommended to use these strains as a
common biocontrol practice in agriculture.
Mishra et al. (2009) tested biocontrol efficacy of five species of Aspergillus and five
species of Trichoderma in vitro against Fusarium oxysporum f. sp. lycopersici. In both
the experiments (dual culture and culture filtrates) T. harzianum was found to be highly
effective against the isolates of Fusarium oxysporum f. sp. Lycopersici (Fol). followed
by A. niger biocontrol potential of A. terreus is least among all the isolates tested.
Culture filtrates obtained from A. luchuensis exerted least inhibition of Fusarium
oxysporum f. sp. Lycopersici (Fol). The most sensitive isolate of Fol. against all the
antagonists tested was identified as IIVR-2 (Fol. 9). Inherent diversity among Fol.
isolates, from different tomato growing regions in India, was determined using RAPD
primers. The genetic similarity coefficients ranged from 0.20 to 0.96, indicating that no
any two or more isolates were 100% similar. RAPD profiles revealed up to 20% genetic
diversity among ten isolates of Fusarium oxysporum f. sp. lycopersici.
Houssien et al. (2010) recommended Biological control and the plant especially when
applied together . Trichoderma harzianum, salicylic acid and low dose of thiophanate
methyl as fungicide as a new strategy to enhance tomato defense response against wilt
disease caused by Fusarium oxysporum f. sp. lycopersici under greenhouse conditions.
Plants were harvested at 35 days after pathogen infection. All applied treatments
completely protected tomato seedlings against Fusarium wilt. Disease index percentage
(DI %) was highly significantly reduced up to zero%. Level of all the determined
physiological parameters greatly changed in tomato plants in response to Fusarium
oxysporum (FO), TH fungi and hormonal elicitor and reflected many components of
defense signals which leading to the activation of power defense system in tomato
against pathogen attack. Application of Trichoderma harzianum and salicylic acid
stimulated all these parameters not only to reach but also exceed their content in healthy
control.
Ramezani (2010) observed the mycoparasitism inhibitory effects of five Trichoderma

species (T. harzianum, T. koningi, T. longiconis, T. hamatum and T. viride) on the
growth of Fusarium oxysporum f.sp. lycopersici .The maximum and minimum
inhibitory effect was shown by T. harzianum and T.viride. In the greenhouse, the
comparison of the efficacy of disease decrease was carried out between soil and seed
treatments affected by T. harzianum spores. Seed treatment did not cause disease
decrease but soil treatment caused disease decrease up to 92%.
John et al. (2010) reported that native isolates of Trichoderma virens (Tv1) increased
the plant growth and highly inhibited the mycelial growth of the pathogen under in vitro
condition. In green house studies seed treatment plus soil application of talc based
formulation of T.virens (Tv1) significantly reduced incidence of the diseases (54.66%
more efficient than control), compared to the other isolates of T.virens. Expression of
various defence related enzymes was found involved in the induction of systemic
resistance against pathogen infection. The enzyme activity increased from 7th day of
sampling and the maximum was observed on 14th day of sampling and then it slightly
decreased.
Srivastava et al. (2010) evaluated a large number of Trichoderma sp. and pseudomonas
isolates for their efficacy to control Fusarium wilt of tomato. T. harzianum was
multiplied on six different substrates out of which Jhangora, an undertilized grain crop,
proved to be the superior substrate. Application of T. harzianum and fluorescent
Pseudomonas by seed bio-priming significantly increased seed germination (22–48%)
and reduced the days required for germination (2.0–2.5 days). All bioagents used in this
study significantly reduced the incidence of wilt in pot and field trials and combinations
of bioagents were more effective than single isolate treatments. The combination of
Pseudomonas fluorescens, T.harzianum and AMF provided significantly better control
than uninoculated treatment, reducing disease incidence and severity by 74% and 67%
in pots and field, respectively. The combination treatments also increased yield by 20%.
Addition of cow dung compost (CDC) further reduced disease and improved yield in all
treatments. Comparing to control (-CDC), the combination of all three bioagents with
CDC significantly reduced disease by 81 and 74% in pots and field, respectively and
enhanced the yield by 33%.
Mwangi et al. (2011) investigated the ability of an isolate of Trichoderma harzianum

(P52) and arbuscular mycorrhizal fungi (AMF) in enhancing growth and control
Fusarium oxysporum f. sp. lycopersici in tomato seedlings. The treatment with both P52
and AMF significantly (P < 0.05) enhanced all growth parameters (heights; shoot and
root dry weight) investigated compared to the control. Disease severity was generally
lower in isolate P52 and AMF fungi treatment either individually or when combined
together, though the effect was not statistically significant (P_ 0.05). A treatment
combination of P52 + AMF had less trend of severity as compared to each individual
fungus.
Ahmed (2011) tested Trichoderma koningii and a white sterile fungus against Fusarium
oxysporum f. sp. lycopersici. Tomato grown in the soil amended with combined inocula
of the individual plant growth promoting fungi (PGPF) and wilt pathogen showed much
a reduced intensity of the disease and promoted growth and yield. The antagonistic
rhizosphere PGPF suppressed the deleterious soil microbes by competing at the active
sites, reduced the intensity of disease development and, subsequently, stimulated the
growth and yield of plants.
Azarmi et al. (2011) evaluated the effect of three Trichoderma isolates including T.
harzianum isolate T969, T. harzianum isolate T447 and Trichoderma sp. isolate T in
tomato seedling vigor and their nutrient uptake via two inoculants introduction
methods.Seed germination rate was not affected by Trichoderma application, but shoot
height, shoot diameter, shoot fresh and dry weight and root fresh and dry weight in
tomato seedlings were interestingly (p ≤ 0.05) increased when sown in Trichoderma sp.
and T. harzianum (T969) fortified soil and when compared to the control. The soil
amended by Trichoderma sp. T and T. harzianum T969 had marked increased in leaf
area (p ≤ 0.05). Chlorophyll content increased in seedling grown in Trichoderma sp. T
amended soil as well as in Trichoderma sp. and T. harzianum T969 coated seed. A
dramatic increase (p ≤ 0.05) in the concentrations of Ca2+, Mg2+, P and K+ were
recorded in the seedling shoot and root among T. harzianum T447 soil amended
treatment when compared to the control, except for Na+ level in soil amendment with T.
harzianum T969 and seed coating with strain Trichoderma sp. T, which significantly
reduced the Na+ concentration.
Alwathnani and Kahkashan (2012) observed that in dual culture technique

Aspergillus niger,Penicillium citrinum, Penicillium sp. and Trichoderma harzianum
inhibited the radial colony growth of the test pathogen. Methanol extract of Nostoc
linckia and Phormidium autumnale showed moderate and minor zone of inhibition.
Maximum seed germination was observed in seeds treated with N. linckia (93%),
whereas, T. harzianum, P. autumnale, P. citrinum showed 80% seed germination, while
A. niger treated seeds showed 50% germination. Under pot conditions, the plant heights,
fresh and dry weight of plants were found to be increased significantly (p ≤ 0.05) in all
treatments except in P. autumnale amended soil. Similar results were observed in
chlorophyll (a+b) content of treated plants. Maximum control of wilt disease was
observed with T. harzianum (44.4%) treated plants as compared to FOL inoculated
plants. Whereas, effectiveness of the other antagonists were recorded in the following
order: A. niger (35.6%), N. linckia (33.3%), P. citrinum (24.4%), and P. autumnale (0.9
%).
Bokhari and Kahkashan (2012) Trichoderma spp. are used in reasonably large
quantities in plant agriculture both for disease control and increased yield. In vitro test
showed that T. harzianum exhibited higher antagonist activity than T. viride against F.
solani. Dual culture and volatile metabolites assays showed that T. harzianum was more
effective in suppressing the growth (51.4 and 38.1%) of pathogen. Culture filtrates of T.
harzianum and T. viride caused reduction in the growth of F. solani by 21.3 and 17.3%,
respectively. Under pot conditions, tomato plants treated with T. harzianum showed
117.5 and 138.9% increase in plant height and dry weight, respectively compared to the
control. T. harzianum caused 55.5% reduction in disease incidence while T. viride
reduced root rot by 41.1%. The results obtained here clearly indicate that T. harzianum
is more effective antagonist against F. solani as compared to T. viride.
Abed et al. (2013) investigated efficacy of Trichoderma spp. neem products and
carbendazim against Fusarium oxysporum f.sp. lycopersici and growth parameter of
tomato plants in pot condition. All the treatments were significantly reduced disease
severity of Fusarium oxysporum f.sp. lycopersici as compared to control. Significantly
reduced percent disease sevirity was recorded in treatment carbendazim (16.9%)
followed by T. harzianum (18.10%), T. viride (19.50%), neem leave extract (20.70%)
and neem cake powder (22.09%) including with control pots (26.01%). All treatments
significantly increased plant height, root length, fresh and dry shoot-root weight as
compared to control.
Sundaramoorthy and Balabaskar (2013) evaluated the efficacy of the native isolates
of Trichoderma species to promote the growth and yield parameters of tomato and to
manage Fusarium wilt disease under in vitro and in vivo conditions. The dominant
pathogen, which causes Fusarium wilt of tomato, was isolated and identified as
Fusarium oxysporum f. sp. lycopersici (FOL). Fifteen native Trichoderma antagonists
were isolated from healthy tomato rhizosphere soil in different geographical regions.
Under in vitro conditions, the results revealed that T. harzianum (ANR-1) isolate was
found to effectively inhibit the radial mycelial growth of the pathogen (by 53%) when
compared to all other isolates. Under greenhouse conditions, the application of
Trichoderma harzianum (ANR-1) exhibited the least disease incidence (by 15.33%).
Also tomato plants treated with Trichoderma harzianum (ANR-1) isolate showed a
significant stimulatory effect on plant height (by 73.62 cm) and increased the dry
weight (by 288.38 g) of tomato plants in comparison to other isolates and untreated
control.
Pseudomonas fluorescens
Duijff et al. (1998) reported that the suppression of fusarium wilt by P. fluorescens
WCS417r was ascribed to systemic induced resistance without any detection of the PR-
proteins tested (PR-1 and chitinases). In contrast, the suppression achieved by
nonpathogenic F. oxysporum Fo 47 appeared to be mainly ascribed to microbial
antagonism but also to a lesser extent to systemic induced resistance. This induced
resistance could be related to the accumulation of PR-1 and chitinases.
Dekkers et al. (2000) reported that pathogenic fungus Fusarium oxysporum f. sp.
radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient
root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a
biocontrol strain. The mechanism for disease suppression most likely is induced
systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391,
which acts through the production of the antibiotic phenazine-1- carboxamide, were
differentially labeled using genes encoding auto fluorescent proteins.
Monda (2002) reported that Fusarium oxysporum f.sp. lycopersici leads to high losses
of tomatoes in many countries. Increasing restraints on the use of pesticides encourages
adoption of use of alternative strategies of controlling the disease. Alternative strategies
include use of biocontrol agents. Bacterial biocontrol agents with promising biocontrol
activities against F. oxysporum f. sp. lycopersici include Pseudomonas flourescens, P.
putida, P. chlororaphis, P. alcaligenes, Bacillus subtilis, Streptomyces pulcher, S.
canascens, S. citreoflourescens, S. corchorusii and S. mutabilis. Fungal biocontrol
agents that are efficacious against F. oxysporum f.sp. lycopersici include Penicillium
oxalicum, P. purpurogenum, Trichoderma harzianum, T. piluliferum, Pythium
oligandrum, non-pathogenic Fusarium oxysporum Fo 47 and Trametes versicolor.
Biological control agents including bacteria and fungi have shown some promise for the
control of Fusarium wilt of tomato.
Ramamoorthy et al. (2002) were of the view that Pseudomonas fluorescens protected
tomato plants from Fusarium oxysporum f.sp. lycopersici. Phenolics were found to
accumulate in bacterized tomato root tissues challenged with F. oxysporum f. sp.
lycopersici at one day after pathogen challenge. They suggested that induction of
defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics
and PR-proteins might have contributed to restriction of invasion of F. oxysporum f. sp.
lycopersici in tomato roots.
Akköprü and Demir (2005) studied the effect of Glomus intraradices Schenck &
Smith and four rhizobacteria (RB; 58/1 and D/2: Pseudomonas fluorescens biovar II;
17: P. putida; 21: Enterobacter cloacae) on tomato with respect to morphological
parameters (fresh and dry root weight) and phosphorous (P) concentration in the roots.
Treatments with single and dual inoculation with G. intraradices and RB strains
reduced disease severity by 8.6–58.6%. Individual bacteria inoculations were more
effective than both the single AMF and dual (G. intraradices + RB) inoculations. In
addition, the RB and G. intraradices enhanced dry root weight effectively. Significant
increases in root weights were recorded particularly in the triple inoculations compared
with single or dual inoculations. Compared with the non-treated controls all biological
control agents increased P-content of treated roots of plants. Colonization with RB
increased especially in triple (FOL + G. intraradices + RB) inoculations whereas
colonization of G. intraradices was significantly decreased in treatment of FOL + G.
intraradices compared with triple inoculations.
Haas and Genevieve (2005) reported that particular bacterial strains in certain natural
environments prevent infectious diseases of plant roots. How these bacteria achieve this
protection from pathogenic fungi has been analysed in detail in biocontrol strains of
fluorescent pseudomonads. During root colonization, these bacteria produce antifungal
antibiotics, elicit induced systemic resistance in the host plant or interfere specifically
with fungal pathogenicity factors. Before engaging in these activities, biocontrol
bacteria go through several regulatory processes at the transcriptional and post-
transcriptional levels.
Kamilova et al. (2006) studied the pathogenic fungus Fusarium oxysporum f. sp.
radicis-lycopersici and of the bacterial biocontrol strain Pseudomonas fluorescens
WCS365, and of both microbes, on the amounts and composition of root exudate
components of tomato plants grown in a gnotobiotic stone wool substrate system.
Conditions were selected under which introduction of F. oxysporum f.sp. radicis-
lycopersici caused severe foot and root rot, whereas inoculation of the seed with P.
fluorescens WCS365 decreased the percentage of diseased plants from 96 to 7%.
Analysis of root exudates revealed that the presence of F. oxysporum f. sp. radicis
lycopersici did not alter the total amount of organic acids, but that the amount of citric
acid decreased and succinic acid increased as compared with the non treated control. In
contrast, in the presence of the P. fluorescens biocontrol strain WCS365, the total
amount of organic acid increased, mainly due to a strong increase of the amount of
citric acid, whereas the amount of succinic acid decreased dramatically. Under
biocontrol conditions, when both microbes are present, the content of succinic acid
decreased and the level of citric acid was similar to that in the non treated control. The
amount of sugar was approximately half that of the control sample when either one of
the microbes was present alone or when both were present. Analysis of the interactions
between the two microbes grown together in sterile tomato root exudate showed that
WCS365 inhibited multiplication of F. oxysporum f.sp. radicis- lycopersici, whereas the
fungus did not affect the number of CFU of the bacterium.
Yigit and Murat (2007) treated tomato roots with biomass of Pseudomonas flourescens
strain from rhizosphere, non-pathogen Fuasrium sp. and Trichoderma harzianum T-22
individually and in combination and planted in artificially infested soil with pathogen F.
oxysporum f. sp. lycopersici. Although all biocontrol agents applied individually
reduced disease incidence, treatments as combination, except for nonpathogen
Fusarium strain + T. harzianum T-22, showed more protective effect. Combination of
T. harzianum T-22 + fluorescent Pseudomonas isolate gave the best control (70.2%).
Rini and Sulochana (2007) evaluated the efficacy of twenty-six local isolates of
Trichoderma spp. and 56 isolates of fluorescent Pseudomonads from Kerala for their
antagonistic activity against Rizoctonia solani and F. oxysporum under in vitro
conditions. Different isolates showed varying degrees of antagonism. The two most
antagonistic isolates against R.solani were T. pseudokoningii TR17 and T. harzianum
TR20. Likewise, T. viride TR19 and TR22 formed the most effective isolates against F.
oxysporum. Production of volatile and non-volatile antibiotic compounds varied among
these isolates of the fluorescent Pseudomonads, Pseudomonas fluorescens isolates P28
and P51 showed the greatest inhibition against R. solani whereas against F. oxysporum,
P20 and P28 were most effective. Isolates obtained from the phylloplane were generally
unsuccessful. Inhibitory property of the antagonistic bacteria was also media-dependent.
Many of the pseudomonads, which inhibited the pathogens on KMB agar, failed to
retard the pathogen’s growth on the PDA medium. The bacterial and fungal antagonists
were also not mutually antagonistic as their co-inoculation hardly inhibited each other.
Srinivasan et al. (2009) evaluated bacteria strains (Pseudomonas putida FC-6B,

Pseudomonas sp. FC-7B, Pseudomonas putida FC-8B, Pseudomonas sp. FC-9B and
Pseudomonas sp. FC-24B) together with Achromobacter sp. AM1 and Serratia sp.
DM1 against Fusarium wilt of tomato. Two commercial bioproducts, Trichoderma
harzianum T22 (Root Shield) and Pseudomonas chlororaphis MA 342 (Cedomon) were
also used, their current study showed the potential biocontrol activity of bacterial strains
FC-6B, FC-7B, FC-8B, FC-9B and FC-24B isolated from re-used rock wool soilless
substrates against Fusarium wilt disease, and the growth promoting activity of these
strains on tomato plants.
Karkachi et al. (2010) evaluated of the antagonistic activity of three bacterial and a
fungi with direct confrontation method and the filtrates culture against the growth
of Fusarium oxysporum f.sp. lycopersici. They observed inhibition of the mycelial
growth of Fusarium oxysporum f.sp.lycopersici with Bacillus cereus energized the low
activity and it was more significant with Serratia marcescens and Trichoderma
harzianum for the 2nd day but with Pseudomonas fluorescens, it was for the 5th day.
The filtrates of culture of these antagonists showed that only Serratia
marcescens and Trichoderma sp. have a rate of inhibition which varies between (40-
95%) and of (20-30%) with Pseudomonas fluorescens.
Asha et al. (2011) evaluated Pseudomonas fluorescens for their efficacy in increasing
seed quality variables of tomato and in inhibiting the mycelial growth of Fusarium
oxysporum. Pseudomonas isolate 2 produced effective results and was selected and
mass multiplied. Fresh cultures of Pf2 isolate was found to increase seedling emergence
and reduce Fusarium wilt disease incidence when compared to the control and the
formulations.
Seleim et al. (2011) reported the isolate of Ralstonia solanacearum wilted roots were
pathogenic to tomato plants and produced typical symptoms of wilt. Isolate No. 5
exhibited the highest virulence followed by isolate No. 4. Pseudomonas fluorescens
, P. putida, Bacillus subtilis and Enterobacter aerogenes were isolated from tomato
rhizosphere and tested against R. solanacearum in vitro and in vivo. All PGPR strains
except E. aerogenes, increased seed germination up to 15% over untreated control.
Under greenhouse conditions, P. fluorescens exhibited the highest disease reduction of
tomato bacterial wilt disease followed by P. putida and then B. subtilis while E.
aerogenes showed the lowest disease reduction percentage. Also tomato plants treated
with all PGPR isolates significantly stimulated plant growth promotion under
greenhouse conditions. In field trails, addition of P. fluorescens, P. putida and B.
subtilis at concentration of 108 cfu mL-1 showed highest disease reduction percentage
in Pseudomonas fluorescens of tomato bacterial wilt disease.
Ardebili et al. (2011) evaluated Pseudomonas fluorescens CHA0 against Fusarium

oxysporum f. sp. lycopersici. Seed bacterization resulted in significantly higher vigour
index of seedlings, indicating growth promoting effects of Pseudomonas fluorescens
CHA0. The presence of bacteria in culture medium inhibited the fungal growth to about
50% in comparison with the control plates. Studies on induction of defense mechanisms
. P. fluorescens CHA0 inoculation significantly induced peroxidase (POX),
polyphenoloxisae (PPO) and superoxid dismutase (SOD) activities in root tissues
whereas phenylalanine amonialyase (PAL) activity and lignification rates remained
unchanged.
Meenakshisundaram and Santhaguru (2012) screened fluorescent Pseudomonas spp.

were for their antagonistic activities against Fusarium solani. They were tested for
phosphate solubilization ability and production of siderophores, hydrolytic enzymes,
indole 3-acetic acid, hydrogen cyanid, Innate antibiotic resistance, and antibacterial
activity of fluorescent pseudomonas against ten target bacterial pathogens of health
significance like Staphylococcus aureus, Streptococcus mutans, Bacillus subtilis,
Aeromonas hydrophila, Vibrio cholerae, Escherichia coli, Klebsiella pneumoniae,
Shigella sonnei, Salmonella typhi and Serratia marcescens by in vitro techniques.
Growth chamber studies resulted in statistically significant increases in plant stand
(29%) as well as in root dry weight (58%). Pf5 was able to establish itself and survive in
tomato rhizosphere after 40 days, following planting of bacterized seeds. Pf5 is a
potential biological control agent that may contribute to the protection of tomato plants
against damping-off caused by F.oxysporum.
Rocha and Andréa (2013) selected six rhizobacteria isolates in controlling Ralstonia
solanacearum and Fusarium oxysporum f. sp. lycopersici (FOL) under controlled
conditions in a greenhouse, and to link such capacity to the "in vitro" production of
selected biologically active compounds. The ability of these rhizobacteria isolates to
produce chitinases, amylases, lipases, antibiotic compounds was investigated.
Additionally, their ability in solubilizing phosphate was also checked. Microbiolization
of seeds with one rhizobacterium DFs1421 (Pseudomonas sp.) reduced the tomato wilt
AUDPC in both assays (36.6 and 91.7% in the first and second assays respectively).
Such efficacy in wilt control is likely to be linked with the production of antibiotics as
observed "in vitro". Streptomyces (DFs1296 and DFs1315) and Bacillus (DFs1414), and
the chemical inducer (ASM) reduced significantly fusarium wilt ranging 22.5 to 76%.
This may be owing to the observed chitinolytic activity and / or antibiosis in the
presence of volatile compounds.
Saravanan et al. (2013) focused on the antagonistic potential of fluorescent

Pseudomonas in vitro, and its inoculation effect on growth performance of Fusarium
oxysporum and Rhizoctonia solani infested soil. Biochemical characteristics of
fluorescent Pseudomonas showed that all ten isolates were positive to catalase, amylase,
gelatinase and siderophore production. While three isolates (Pf5, Pf6 and Pf9) were
oxidase positive, nine isolates (Pf1, Pf2, Pf3, Pf4, Pf6, Pf7, Pf8, Pf9, and Pf10) were
tolerant to 6.5% NaCl. Isolates Pf5 and Pf6 were resistant to all the test antibiotics; in
contrast, the remaining eight isolates responded differently to different antibiotics.
Isolates Pf5 and Pf6 were antagonistic against 14 bacterial species, and two pathogenic
fungi (F. oxysporum and R. solani). Inoculation with fluorescent Pseudomonas Pf5
induced a significant increase in root and shoot length, and dry weight. Treatment of
plants with either F. oxysporum or R. solani drastically reduced the root and shoot
length and dry weight of the plant. However, in the presence of fluorescent
Pseudomonas the adverse effect of the pathogens on growth of L. esculentum was
alleviated.
Tayal et al. (2011) investigated the effectiveness of Glomus fasciculatum, Trichoderma

viride and Pseudomonas fluorescens, alone and in combinations to control Fusarium
oxysporum f.sp. lycopersici. The three biological control agents were effective in
controlling the disease; however the success rate for inhibition varied among the
different treatments. Plants inoculated with P. fluorescens had higher concentration of
phenol and greater activities of phenylalanine ammonia lyase and catalase. Inoculation
with T. viride led to maximum induction of anti-oxidative enzymes such as catalase and
peroxidase. On the other hand, G. fasciculatum-inoculated plants showed improved
growth and highest phosphorus uptake. A combination of all the three biocontrol agents
together, promoted growth and inhibited disease up to 94% in tomato plants. Thus, use
of multiple biocontrol agents leads to enhanced level of disease resistance than
individual use of bio-inoculants through the induction of multiple defense mechanisms.
Soil solarization
Martyn and Hartz (1986) reported that soil solarization for either 30 or 60 days was
effective in delaying the onset of wilt symptoms as well as in reducing total disease
incidence in a Fusarium-susceptible watermelon cultivar, Sugarbaby, but complete
disease control was not achieved. These effects lasted over two growing seasons;
however, best control was obtained during the first year. Temperature maxima during
17-19 July 1984 in solarized soil were 60, 50, 42, and 37 C at depths of 2, 10, 20, and
30 cm, respectively. The population of Fusarium oxysporum f.sp. niveum was reduced
dramatically throughout the soil profile after 30 days of solarization compared with a
similarly infested, nonsolarized treatment. Further decline in F. oxysporum f. sp. niveum
was achieved in the top 10 cm of soil after the 60-day solarization. Populations of
saprophytic Fusarium spp. were two fold higher at 15-20 cm deep and eight fold higher
at 30-35 cm deep after 30 days of solarized than in non-solarized soil.
Overman and Jones (1986) solarized Eau GaIlie fine sand and compared it to a
Sesbania macrocarpa Muhl. cover cropped area. Both areas were adjusted to pH 6.0 or
7.5, further divided by soil fumigant treatments [none; methyl isothiocynate 17% + 1,3
clichloropropene 34% + chloropicrin 15% (V-201) 35 gal/acre; or methyl bromide 98%
+ chloropicrin 2% (MBC) 300 lb./acre, transplanted with 'Sunny’ tomato (Lycopersicon
osculonrum Mill.) in the fall 1985 and replanted with ’Sunny’ tomato in the spring 1986
without disrupting the mulched bed. Solarization reduced the incidence of Verticillium
alba-atrum race 2 Reinke & Berth through both crop seasons. Tomato yield was
improved by solarization 26% in the fall but not affected in the spring. The higher pH
level controlled Fusarium oxysporum Schlecht. f.sp. Iycopersici (Sacc.) Snyd. & Hans.
Race 3 through both crops and increased the spring yield 32%. Both fumigants
improved yield only in nonsolarized blocks, MBC controlled Fusarium wilt also in the
spring double-crop. Highest yields occurred in plots which were solarized in the off-
season and fumigated in the fall.
Oliveira (1992) reported that soil solarization was feasible method and more
advantageous than the traditional soil disinfestations using steaming or fumigants.
Fumigants are hazardous and both methods are expensive and detrimental to the
biological equilibrium in the soil. Furthermore, re-infestations occur very often and
heavy losses of crops are subsequently observed.
Chellemi et al. (1997) reported that soil solarization was shown to be cost effective,
compatible with other pest management tactics, readily integrated into standard
production systems, and a valid alternative to preplant fumigation with methyl bromide
under the tested conditions. Solarization using clear, photoselective, or gas-
impermeable plastic was evaluated in combination with methan sodium, 1, 3-
dichloropropene + chloropicrin, methyl bromide + chloropicrin, pebulate, or cabbage
residue. The severity of root galling was lower (P < 0.05) when soil solarization was
combined with 1, 3-dichloropropene + chloropicrin (16.2 + 3.4 g/m2) and a gas-
impermeable film. The incidence of bacterial wilt was not affected by soil treatments.
Marketable yields in plots using various combinations of soil solarization and other
tactics were similar (P < 0.05) to yields obtained in plots fumigated with methyl
bromide + chloropicrin.
Stapleton (2000) reported that Solarization occurs through a combination of physical,

chemical, and biological mechanisms, and is compatible with many other disinfestations
methods to provide integrated pest management. Commercially, it is used on a
relatively small scale worldwide as a substitute for synthetic chemical toxicants, but its
use is increasing as methyl bromide, the major chemical fumigant, is phased out due to
its ozone-depleting properties. Solarization currently is an important and widespread
practice for home gardeners.
Matheron and Martin (2010) initiated studies to assess the potential of summer soil
solarization and flooding as management tools for Fusarium wilt of lettuce in
southwestern Arizona production fields. Soil naturally infested with F. oxysporum f. sp.
Lactucae that was solarized from 2 to 8 weeks was consistently greater than growth in
nonsolarized soil. Growth of lettuce in flooded soil containing the pathogen
occasionally was significantly higher than in no flooded soil; however, the effect on
plant growth and health was not as consistent as that recorded for solarized soil. The
incidence of Fusarium wilt after solarization was reduced from 42 to 91% compared
with disease in nonsolarized plots. There was no significant benefit of a 2- over a 1-
month solarization period under the conditions of these trials, where the mean soil
temperature at a depth of 5 cm during a 1-month solarization period in 2005 and 2006
was 47 and 49°C.
Barakat and Mohammad (2012) carried out soil solarization against Fusarium
oxysporum f. sp. lycopersici, for seven weeks through July and August 2008 and 2009
in the climatic conditions of Al-Aroub Agricultural Experimental Station, located in the
southern mountains of the West Bank, Palestine. Double polyethylene (DPE) sheets,
regular polyethylene (PE) sheets, and virtually impermeable films (VIF) were compared
to examine their effects on soil temperature, disease severity, and plant growth. Results
showed that in comparison to the control, PE, DPE, and VIF treatments increased the
mean maximum soil temperatures by 10.2, 14.1, and 8.8°C, respectively, in 2008 and by
10.2, 12.6, and 8.3°C respectively, in 2009. The longest length of time recorded for
temperature above 45°C under DPE sheets were 220 hours in 2008 and 218 hours in
2009. The treatments reduced the pathogen population by 86% and the disease by 43%
under the DPE treatment in 2009 and to a lesser extent by the other treatments.
Increases of up to 94% in fresh plant weight and up to 60% in plant dry weight were
evident under the same treatment. The treatments also increased soil organic matter.
Arora et al. (2013) are of the view that Soil-solarization, a hydrothermal process, is
very simple, cheap and nonhazardous technique, to cover airtight the moist soil with
polyethylene mulches/sheeting during the period of intense solar radiations for the
particular duration results in increases in the soil temperature. In the present
investigations, plots for raising nurseries of vegetable crops were mulched with thin
transparent polyethylene sheets for 8 weeks. During Solarization, the temperature on an
average was 10 to 12°C higher than the unmulched soil. The plot (size-1m²) before
mulching with transparent polythene sheets amended with organic amendments farm
yard manure (2 kg/plot), poultry manure (2 kg/plot)) and bioagents Trichoderma
harzianum (6 gm/plot), Pseudomonas fluorescens (6 gm/plot)) further increase soil’s
suppressivity potential. The microbial population existing in soil was modified due to
the hydrothermal process. Total populations of fungi, bacteria, actinomycetes,
Trichoderma spp. except Pseudomonas fluorescens decreased drastically and
significantly due to the solarization for 8 weeks. The microbiological changes of
naturally existing populations of fungi, bacteria, actinomycetes, Trichoderma spp. were
decreased and of P. fluorescens were increased by solarization carried out for 8 weeks.
However, after 30 days after raising a nursery crop, the estimated population showed
significant recovery except the population of P. fluorescens, changed only marginally.
Carbendazim
Rajput et al. (2006) conducted pathogenicity test of Fusarium oxysporum f. sp.

vasinfectum on four cotton varieties viz, Cris-9, TH-41/83. CIM-448 and Shahbaz- 95.
The growth of artificially inoculated plants of Cris-9 and TH-41/83 significantly
reduced and plants were completely dried after 20 days of inoculation. The numbers of
germinated plants were also lower as compared to Shahbaz-95 and CIM-448.
Carbendazim at its three doses significantly inhibited the mycelial growth of the fungus
followed by Thiophanate methyl and Dithane M-45. There was no significant difference
in mycelial growth of the fungus on Thiovit (35.75 mm) and control (37.87 mm) in
which fungicide was not used. The maximum root and shoot length of cotton varieties
was recorded with Carbendazim followed by Thiophanate methyl. The growth was
significantly increased in Shahbaz-95 and CIM-448 as compared to Cris-9 and TH-
41/83 varieties. Carbendazim also significantly reduced the colonization of Fusarium
oxysporum f. sp. vasinfectum on root pieces of Cris-9 variety. The growth of Cris-9 also
increased when plants were inoculated with low level of inoculum (10.75cm and
12.25cm) than at high inoculum levels (6.72cm and 5.85cm).
Omar et al. (2006) evaluated Bacillus megaterium (c96) and Burkholderia cepacia
(c91) against Fusarium oxysporum f.sp. radicis-lycopersici as biocontrol agents alone
and when integrated with the fungicide carbendazim. In an initial screening, these
isolates reduced disease incidence by 75 and 88%, respectively. In vitro, both biocontrol
agents were highly tolerant to the fungicide carbendazim, commonly used to control
fusarium diseases. Carbendazim reduced disease symptoms by over 50% when used at
> 50 μ g mL – 1 , but had little effect at lower concentrations. Combination of the
bacterial isolates and carbendazim gave significant (P ≤ 0·05) control of the disease
when plants were artificially inoculated with the pathogen. Application of carbendazim
at a low concentration (1 μ g mL – 1 ) in combination with B. cepacia c91 reduced
disease symptoms by 46%, compared with a reduction of 20% obtained with the
bacterium alone. A combination of B. megaterium c96 with an increased application
rate of 10μg mL − 1 carbendazim significantly reduced disease symptoms by 84%
compared with inoculated controls and by 77% compared with carbendazim treatment
alone.
Amini and Dzhalilov (2010) evaluated six fungicides; benomyl, carbendazim,

prochloraz, fludioxonil, bromuconazole and azoxystrobin, for their efficacy against
Fusarium oxysporum f. sp. lycopersici in vitro and in vivo. Seven different
concentrations against the pathogen inhibitory activities through mycelial growth
inhibition on potato media. Four concentrations of above mentioned fungicides (0.1, 1.
10 and 100 μg/ml) were tested for controlling Fusarium wilt on tomato plants in
glasshouse. Fungal radial growth was measured and median effective concentration
(EC50) values (μg/ml) determined. The result of glasshouse tests revealed a different
degree of efficacy of all tested fungicides in reducing disease infestation. Prochloraz
and bromuconazole were the most effective fungicides against the pathogen both in
vitro and in vivo, followed by benomyl and carbendazim.
Singha et al. (2011) evaluated the effectiveness of crude chloroform extract of Piper
betle (PbC) against Fusarium oxysporum f.sp. lycopersici. It was observed that 1%
(w/w) amendment of the PbC in soil was more efficient in reducing the Fusarium
population in soil than carbendazim and the combined amendment of carbendazim and
PbC. Variation in different parameters like shoot growth, root growth and mean fresh
weights of tomato seedlings in all the treatments were recorded. Higher accumulation of
total phenolics was observed in the Fusarium-infested plants as compared to that of
healthy control and PbC-treated plants. Moreover, it was observed that the extract could
reduce the symptoms and disease development.
Ajay and Shashi (2012) are of the view that tomato wilt is a dreaded disease of
vegetable in the warm humid subtropical and temperate regions in the world and thus
cause heavy losses (10%~90%) in yield. The effective control of wilt can be done by
seed treatment with Thiram 75 WDP before sowing followed by 10 minute dipping of
seedlings roots in 0.3% solution of Carbendazim 50 WP before transplanting and plant
roots drenched with Copper oxychloride 50 WP @ 0.3 % solution+0.01 % Streptomycin
solution one month after transplanting.
Chapter - III
MATERIALS AND METHODS
L
3.1. Experimental site:
The present study entitled “Integrated Disease Management against Fusarium

oxysporum f.sp. lycopersici on Tomato (Lycopersicon esculentum L.)’’was carried
out in the Bio-net house, Department of Plant Pathology, Sam Higginbottom Institute of
Agriculture, Technology & Sciences, Naini, Allahabad 2013-2014.
3.2. Topographical conditions:
Allahabad district of Uttar Pradesh, India is situated at 25.27º north and 81.50º
east latitude with an altitude of 98 m above the mean sea level. The climate is typically
semi-arid and sub-tropical. The maximum temperature reaches up to 47.5 ºC in the
summers and drops down to 1.5 ºC in the winters. The average rainfall in this area is
about 1100 mm per annum. Most of which generally occur during winter season.
3.3. Cleaning and sterilization of glass wares:
The Petri plates, pipettes conical flasks, test tubes, beakers etc. used in the
experiments were thoroughly washed and dried. The Petri-plates and pipettes were
wrapped in a silver foil and sterilized in hot air oven at 160ºC for 2 hours (Aneja,
2004).
3.4. Preparation of media (PDA):
For isolation of Fusarium oxysporum f.sp. lycopersici from the infected root stock
of tomato in the laboratory ,the procedure for the preparation of medium was adopted
(Aneja, 2004).
Agar 20 gm.
Dextrose 20 gm.
Peeled potato 200gm
Distilled water 1000 ml
pH 6-6.5
AARITEA MLA ALAIRETAM 39

Procedure:
The potatoes were peeled and cut into small pieces and boiled in 500 ml of water
till they become soft. The extract obtained was filtered through cheese cloth and all the
liquid was squeezed in beaker. Twenty gram agar was added bit by bit to the rest of 500
ml hot water to dissolve. Then 20 g of dextrose was added. Volume of agar and broth
made up to 1000 ml by adding more distilled water. Then 200 ml of the medium was
dispensed to each of five conical flasks and sterilized at 121.6 ºC at 15 lbs
pressure/inch2 for 20 minutes in an autoclave (Aneja, 2004).
3.5. Preparation of Czapek's media (CDA):
For re-isolation of Fusarium oxysporum f.sp. lycopersici from mother culture in

the laboratory the Czapek's Dox Agar medium were used.
Composition
Sucrose 30
Sodium nitrate 2
Dipotassium phosphate 1
Magnesium sulphate 0.5
Potassium chloride 0.5
Ferrous sulphate 0.01
Agar 15
Distilled water 1000 ml
pH 5.5- 6
Preparation of (CDA) :
All the ingredients except agar were suspended, then 15 g of agar was added bit
by bit till dissolved in 1000 ml distilled water. Heat to boiling to dissolve the medium
completely. Sterilize the medium by autoclaving at 15 lbs pressure (121.6°C) for 15
minutes (Eaton et al. 1998).

3.6. Isolation and purification of Fusarium oxysporum f.sp. lycopersici:
Fusarium oxysporum f.sp. lycopersici were isolated from infected roots of tomato
by tissue segment method. The infected roots of tomato were cut separately into small
pieces into the wash glass with the help of sterile blade and surface sterilized with
mercuric chloride (0.1%) for 20-30 seconds followed by three times rinsing with
sterilized distilled water. The diseased pieces were then placed on pre-sterilized blotting
paper to remove excess moisture. The surface sterilized diseased pieces were then
aseptically, transferred on PDA medium and incubated at 28±2˚C for five days. After
incubation, colonies were observed and identified on the basis of morphological and
reproductive characters. The pure cultures were maintained on Czapek's Dox Agar
medium and preserved at low temperature in refrigerator for use as and when required
(Plate 3.1,3.2 and 3.3) (Raithak and Gachande , 2013).
Plate: 3.1 Isolation and purification of Fusarium oxysporum f.sp. lycopersici

Plate: 3.2 Pure culture of Fusarium oxysporum f.sp. lycopersici on PDA medium
Plate: 3.3 Pure culture of Fusarium oxysporum f.sp. lycopersici on Czapek's

medium

3.7. Characters of Fusarium oxysporum f. sp. lycopersici (Sacc.):
The mycelia of Fusarium oxysporum f.sp. lycopersici (Sacc.) W.C. Snyder and
H.N. Hans are delicate white to pink, often with purple tinge, and are sparse to
abundant. The fungus produces three types of spores: microconidia, macroconidia
(Plate 3.4), and chlamydospores. Microconidia are borne on simple phialides arising
laterally and are abundant, oval-ellipsoid, straight to curved, 5-12 x 2.2-3.5 mm, and
nonseptate. Macroconidia, sparse to abundant, are borne on branched conidiophores or
on the surface of sporodochia and are thin walled, three- to five-septate, fusoid-subulate
and pointed at both ends, have pedicellate base. Three-septate conidia measure 27-46 x
3-5 mm while five-septate conidia measure 35-60 x 3-5 mm. Three-septate spores are
more common. Chlamydospores, both smooth and rough walled, are abundant and
form terminally or on an intercalary basis. They are generally solitary, but occasionally
form in pairs or chains. No perfect stage is known.
Plate: 3.4 Microscopic view of conidia (micro and macro) of Fusarium oxysporum
f.sp. lycopersici (40 ×)
3.8. Classification of Fusarium oxysporum f.sp. lycopersici :
Kingdom - Mycota
Division - Eumycota

Sub division - Deuteromycotina
Class - Hyphomycetes
Order - Tuberculariales
Family - Tuberculariaceae
Genus - Fusarium
Species - oxysporum
f.sp - lycopersici
(Ainsworth et al., 1973)
3.9. Counting c.f.u. of Fusarium oxysporum f.sp. lycopersici per one

gram of mycelium:
One gram of Fusarium oxysporum mycelium was weighed from ten days old
Fusarium oxysporum f. sp. lycopersici actively growing culture and the volume was
made up to 10 ml with sterilized distilled water and was shaken well and makes dilution
of further 1:10. 1 ml suspension was taken out with sterile pipette and transferred to 9
ml of sterilized distilled water in a test tube of dilution 1:100. The same procedure was
followed up to10-3dilution. Transfered 1 ml from 10-3 dilution into 3 sterile petri dishes.
fifteen ml of lukewarm PDA medium was poured to each Petri plates. After
solidification of the media, all the plates were incubated in an inverted position at 25+
20C for 3 days into the BOD chamber. After 3 days average numbers of colonies of
Fusarium oxysporum were counted in each plate. The c.f.u.of Fusarium oxysporum f.
3
sp. lycopersici was found to be 14× 10 cfu/gm.
The number of colony forming units (c.f.u.) present in 1g of mycelium was calculated
by the following formula (Aneja, 2004).
Number of colonies
Organism per ml/gm of the sample = ------------------------------------
Amount plated × dilution factor

3.10. Counting c.f.u of Fusarium oxysporum f.sp. lycopersici per one
gram of soil :
Ten gram of mixture of soil that infested with pathogen was weighed and the
volume was made up to 100 ml with sterilized distilled water and was shaken well
(1:10). Out of this suspension 1 ml was taken out with sterile pipette and transferred to 9
ml of sterilized distilled water in a test tube (1:100). Out of this suspension 1 ml was
taken out with sterile pipette and transferred to 9 ml of sterilized distilled water in a test
tube (1:1000). Transfered 1 ml from 10-3 into 3 sterile Petri dishes. Poured 15 ml of
lukewarm PDA medium. After solidification of the media, incubated all the plates in an
inverted position at 25+ 20C for 3 days. After 3 days average number of colonies was
calculated per plate of Fusarium oxysporum. The c.f.u. was found to be 3× 105 cfu/gm .
The number of colony forming units (c.f.u.) present in 1g of soil was calculated by the
following formula (Aneja, 2004).
Number of colonies
Organism per ml/gm of the soil = -------------------------------------------
Amount plated × dilution factor
3.11. Mass multiplication of Fusarium oxysporum f.sp. lycopersici on

Sorghum (Sorghum bicolor L. moench) to prepare sick microplots:
The sorghum grains were soaked partially for one hour in warm water (40 to 45
C) and then spread on the clean blotting paper for air drying. About 150 g moistened
grains were filled in each 250 ml flask with 10 ml water and autoclaved at 15 lbs psi
pressure. The mycelium bit of pure culture of Fusarium oxysporum f. sp. lycopersici
were inoculated under aseptic condition in the flasks containing grains and incubated at
28± 20º C for 10 days. Meanwhile flasks were shaken to avoid clumping of grains and
to facilitate early growth of the fungus. The grains turned whitish due to mycelial
growth of the test fungus. To prepare sick plots of Fusarium oxysporum f. sp.
lycopersici, the grains colonized by Fusarium oxyspoum f.sp. lycopersici were mixed in
the soil (Plate 3.5) (Kamdi et al., 2012) .

Plate 3.5: Mass multiplication of Fusarium oxyspoum f.sp. lycopersici on sorghum
grains
3.12. Preparation of the sick microplot of F. oxysporum f. sp. lycopersici

The inoculum was prepared by growing culture of Fusarium oxysporum f.sp.
lycopersici in a modified liquid broth Czapek's Dox (CD) (Esposito and Fletcher,
1961) on a rotary shaker with indirect fluorescent light at room temperature (22º C).
After 15 days, the contents of the flask were filtered through eight layers of sterile
cheesecloth. One hundred milliliters of the filtrate, which was predominantly
microcondia, was mixed with 4 L of a twice-autoclaved sand / sorghum grains (4:1, v/v)
mixture. The mixture was incubated for 8 weeks at room temperature to allow the
fungus to colonize the medium extensively. To verify that Fusarium oxysporum f.sp.
lycopersici had been established successfully. The infested soil was incorporated into
the top 45 cm of 6 microplots. Microplots were planted with seeds of the wilt-
susceptible tomato cultivar CO - 3. High percentage of wilt had occurred in all infested
plots while no wilt occurred in uninfested two microplots. The remaining aboveground
plant material was cut off at the soil line and removed. On 2nd May 2013, each microplot

was given light irrigation and covered with an ultraviolet stabilized, 30 µm clear
polyethylene film in 4 microplots. The plastic was removed thrice from four microplots
at 15 days interval that is up to June 2013 (Martyn and Hartz, 1986), two microplots
was kept with nonsolarized soil with Fusarium infested.
3.13. Koch's postulate to prove pathogenic potential of Fusarium

oxysporum f.sp. lycopersici:
Fusarium oxysporum f.sp. lycopersici was isolated from infected tomato roots and
stem portions and purified after identification. Pathogenicity of isolate was tested on
healthy tomato seedling at the three-true-leaf stage. Their roots were cut and dipped into
conidial suspension (106 spores/ml) of tested isolate for 30 minute , and then the
seedlings were transplanted into sterilized soils in pots (10 cm in diameter) and kept in
Bio-net. After symptoms appeared the pathogen was re-isolated from infected portion of
tomato seedling and again identified through microscopic in the laboratory (Plate 3.6
and 3.7) (Amini , 2009).
With Fusarium Without Fusarium
l
Plate 3.6: Pathogenicity of Fusarium oxysporum

With Fusarium Without Fusarium
l oxysporum f.sp.
Plate 3.7: Koch's postulate to prove pathogenicity of Fusarium
lycopersici
3.14. Process of soil solarization:
Soil solarization was conducted for 2 months from 15th April to 15th June 2013 at
research field of SHIATS, Allahabad with the following steps:
Step-1: The microplots was thoroughly cultivated and then leveled to prevent the tearing
of polythene sheet.
Step-2: Irrigation was given prior to laying of the polythene sheet.
Step-3: Clear, transparent (not black or colored) polythene sheet of 40 µm thickness was
used , the width of the sheeting was 3 m without any joint
Step-4: The polythene sheeting was done immediately after irrigation.
Step-5: All free edges were buried and the soil around them compacted so as to prevent escape
of heated air or soil moisture (Plates 3.8 and 3.9).

Plate 3.8: Irrigation given prior to laying of the polythene sheet for soil solarization
Plate 3.9: Soil covered with transparent polyethylene sheet for soil solarization

3.15. Source of Tomato seeds:
Tomato cultivar seeds CO - 3 were collected from IIVR (Indian Institute of

Vegetable Research), Varanasi, Uttar Pradesh, India.
3.16. Source of Pseudomonas fluorescens and Trichoderma harzianum
Pseudomonas fluorescens and Trichoderma harzianum were acquired from

Yash Trichoguard , DBT Referral Lab, SHIATS, Allahabad, Uttar Pradesh, India.
3.17. Source of Neem cake and spent mushroom compost
Neem cake and spent mushroom compost acquired from Department of Plant
Pathology, Sam Higginbottom Institute of Agriculture, Technology & Sciences, Uttar
Pradesh, India.
3.18. Application of bio-agents, carbendazim, neem cake and spent

mushroom compost:
The solarized and unsolarized soil was mixed with FYM @ 100 g / pot, the
bioagents Trichoderma harzianum and Pseudomonas fluorescens were applied @ 2 g /
pot, four days before sowing the seeds of tomato variety (CO-3), carbendazim 50 % W.P
was applied @ 2 kg a.i / ha, whereas Neem cake powder was amended in 10 pots with
capacity 10 kg soil / pot @ 10 g/ pot and spent mushroom compost was mixed with soil
in the pots @ 20 g / kg of soil. Seven days after germination, four seedlings per pot
were maintained in each treatment.

3.19. Treatments detail:
Treatments Dosage Nature of References

Treatment
Neem cake 10 g / pot Soil treatment Kimaru et al., 2004
Spent mushroom compost 20 g / kg Soil treatment Raj and Kapoor, 1997
Pseudomonas fluorescens 2 g / pot Soil treatment Simon and Anamika

,2011
Trichoderma harzianum 2 g / pot Soil treatment Simon and Anamika

,2011
Carbendazim 2 kg a.i / ha Soil treatment Van Gestel et al., 2004
3.20. Experimental Details:
Experimental design was laid out in a Complete Randomized Design (CRD) with
three experiments, each experiment with seven treatments and five replications.
3.20.1. Effect of solarized soil with neem cake and carbendazim against
Fusarium oxysporum f.sp. lycopersici of tomato:
The solarized and unsolarized soil was mixed with FYM @ 100 g / pot and was
filled in thirty five pots. Out of thirty five, 20 pots were filled with solarized soil, All the
pots were watered and kept inside the net house. Neem cake powder was amended in 10
pots capacity 10 kg soil / pot @ 10 g/ pot, whereas carbendazim 50 % W.P was applied
@ 2 kg a.i / ha as per ICAR recommendation rate. Ten seeds of tomato variety (CO-3)
were sown per pot, four seedlings per pot were maintained for treatments and untreated
five pots served as control.

Details of treatments
Symbol Treatments
T0 Fusarium oxysporum alone
T1 Solarized soil + Fusarium oxysporum
T2 Solarized soil +Neem cake+Fusarium oxysporum
T3 Solarized soil +Carbendazim + Fusarium oxysporum
T4 Solarized soil + Neem cake+Carbendazim + Fusarium oxysporum
T5 Carbendazim + Fusarium oxysporum
T6 Tomato plant alone
3.20.2. Effect of carbendazim and solarized soil with Pseudomonas

fluorescens, spent mushroom compost against Fusarium oxysporum
f.sp. lycopersici of tomato:
The solarized and unsolarized soil was mixed with FYM @ 100 g / pot and was
filled in thirty five pots. Out of thirty five, 20 pots were filled with solarized soil of the
infested soil. Carbendazim 50 % W.P was applied @ 2 kg a.i / ha, whereas spent
mushroom compost was mixed with soil in the pots @ 20 g / kg of soil and
Pseudomonas fluorescens @ 2 g / pot 4 days before sowing the seeds of tomato variety
(CO-3). Seven days after germination, four seedlings per pot were maintained in each
treatment. Five untreated pots served as control.
Symbol Treatment
T2 Solarized soil + spent mushroom compost +Fusarium oxysporum
T3 Solarized soil +Pseudomonas fluorescens+ Fusarium oxysporum
T4 Solarized soil + spent mushroom compost +Pseudomonas fluorescens +
Fusarium oxysporum

3.20.3. Effect of carbendazim and solarized soil with Trichoderma
harzianum, spent mushroom compost against Fusarium oxysporum
f.sp. lycopersici of tomato:
FYM @ 100 g / pot was mixed with solarized and unsolarized soil and was filled
in thirty five pots. Out of thirty five, 20 pots were filled with solarized soil of the
infested soil. Carbendazim 50 % W.P was applied @ 2 kg a.i / ha, whereas spent
mushroom compost was mixed with soil in the pots @ 20 g / kg of soil and
Trichoderma harzianum @ 2 g / pot 4 days before sowing the seeds of tomato variety
(CO-3). Seven days after germination, four seedlings per pot were maintained in each
treatment. Five untreated pots were served as control.
Symbol Treatment
T2 Solarized soil + spent mushroom compost+Fusarium oxysporum
T3 Solarized soil + Trichoderma harzianum + Fusarium oxysporum
T4 Solarized soil + spent mushroom compost + Trichoderma harzianum +
Fusarium oxysporum
3.21. Layout detail of each experiment:

Experimental design CRD
Crop Tomato
Variety CO - 3
Total No. of treatments 7
Total no. of replications 5
Pot capacity 10 kg soil
Total no. of Pots 35

Seedling /pot 4 seedling / pot
Season Rabi
3.22. Observations recorded:
 Shoot length (cm) at 30, 60, 90, 120 and 150 DAT
 Disease intensity (%) at 60, 90, 120 and 150 DAT
 Root length (cm) 150 DAT
 Fresh weight of shoot (g) 150 DAT
 Dry weight of shoot (g) 161 DAT
 Fresh weight of root (g) 150 DAT
 Dry weight of root (g) 161 DAT
3.23. Measurement of disease intensity % and Reduction %:
The O-4 scale of the disease severity was classified as follows:

0: No wilt symptoms
1: Slight severity, where 25% leave become wilted and one or two leaves became
yellow
2: Moderate severity, two or three leaves became yellow, 50% of leaves became wilted
3: Extensive severity, the all plant leaves became yellow, 75% of leaves become wilted
and growth is inhibited
4: Complete severity, the whole plant leaves become yellow, 100% of leaves become
wilted and the plants die.
The percentage of disease intensity was determined using the formulas as given by
Song et al. (2004).
∑ Scale × number of plants infected
Disease intensity (%) = ------------------------------------------------- ×100

Highest scale × total number of plants
Reduction (%) of disease was calculated by using the following formula according to
(Alwathnani and Kahkashan, 2012).
Control- treatment
Reduction (%) = --------------------------------- ×100
Control

3.24. Statistical Analysis:
In the experiment Complete Randomized Design (CRD) was adopted. The analysis of
variance (ANOVA) technique was applied for drawing conclusion from data. The calculated
values were compared the tabulated values at 5% level of probability (Fisher and Yates,
1968) for the appropriate degree of freedom. The skeleton of analysis at variance table is as
follows:
Table -3.1 Skeleton of the analysis of variance (ANOVA)
Source of Degree of Sum of Mean sum of F(cal) F(tab) at

variance freedom square square 5%
Due to (t-1) SST SSR SST

MSST=
treatment (r  1) MSSE
Due to error T (r-1) SSE SSE

MSST=
t (r  1)
Total Rt-1 TSS
Where,
T= Number of treatments
R= number of replication
SST = Sum of squares due to treatment
MSST= Mean sum of square due to treatment
MSSE = Mean sum of square due to error
TSS= Total sum of square
F.cal = Calculated value of F
F.tab = Value of F from variance ratio table

S. Ed. = Standard error of difference
C. D. = Critical difference
If the calculated value of F is less than table value of F at 5% level of significances

there is no significant difference in the effect of the treatment. However the calculated
value of F is more than table value of F at % level of significance then there is
significant difference in the effect of treatments.
Plate 3.10: Seedlings of Tomato plant

Plate 3.11: Infected tomato plants
Infected Healthy
Plate 3.12: Infected and healthy stems of tomato

Plate 3.13: Removal of extra seedlings
Plate 3.14: View of experimental pots

Chapter - IV
RESULTS AND DISCUSSION
The present study entitled “Integrated Disease Management against Fusarium

oxysporum f.sp. lycopersici on Tomato (Lycopersicon esculentum L.)’’ was
conducted in the green house, Department of Plant Protection, Sam Higginbottom
Institute of Agriculture, Technology & Sciences, Naini, Allahabad in the pots during
rabi season of 2013 - 2014 . The findings of the experiments are presented in the tables
and figures with appropriate headings.
4.1. Effect of solarized soil with Neem cake and carbendazim on plant
growth parameters of tomato plants
Shoot length (cm), at 30 DAT
The results of Table 4.1 and Fig. 4.1 revealed that at 30 days after treatment
solarized soil with neem cake + F.o (T2) and (T4) neem cake with carbendazim+F.o
significantly increased the shoot length of tomato from solarized soil + carbendazim +
F.o (T3) and carbendazim + F.o (T5) .However the treatments of (T2, T4) and (T3, T5)
were shown non-significant among each other. Significantly reduced the length of the
tomato plants were found in T0 (F.o alone) and T1 (solarized soil + F.o) as compared
with other treatments, where as both the treatments (T0, T1) were found non-significant
among each other.

At 60 days after germination the shoot length of tomato was significantly
increased in (T4) solarized soil + neem cake + carbendazim+F.o followed by solarized
soil + carbendazim+F.o (T3), solarized soil + neem cake+F.o (T2) and carbendazim+F.o
(T5) as compared with T0 (F.o alone) and T1 (Solarized soil +F.o). However among the
treatments (T4, T3, T2, T5) and (T0, T1) found non-significant among each other in (Table
4.1 and Fig. 4.1).
RESULTS AND DISCUSSION 59

At 90 days after germination the shoot length of tomato was significantly
increased in solarized soil with tomato plant without F.o (T6) followed by solarized soil
+ carbendazim (T3) as compared with solarized soil + neem cake (T2), solarized soil +
neem cake + carbendazim (T4). However among the treatments (T6, T3) and (T2, T4)
were found non-significant among each other. Significantly reduced the height of
tomato was observed in T0 (F.o alone) followed by T1 (Solarized soil + F.o) were found
non-significant from each other. Among the treatments T5 (carbendazim+F.o) were
significantly increased the shoot length from (T0, T1) but significantly reduced from (T4
T3 T2) in (Table 4.1 and Fig. 4.1).
Shoot length (cm), at 120 and 150 DAT
The results of Table 4.1 and Fig. 4.1 revealed that at 120 and 150 days after
treatment the shoot length of tomato significantly increased in solarized soil with
tomato plant without F.o (T6) followed by solarized soil + carbendazim + F.o (T3),
solarized soil + neem cake+ F.o (T2) , as compared with other treatments , Among the
treatments T4 (solarized soil + neem cake + carbendazim+ F.o) and T5 (carbendazim+
F.o) were found non-significant from each other but significantly increased the shoot
length as compared with T0 (F.o alone) and T1 (Solarized soil + F.o) however the
treatments of (T0, T1), (T2, T3) and (T4, T5) were not significant from each other in
(Table 4.1 and Fig. 4.1).
Fresh shoot weight (g), at 150 DAT
At 150 days after germination the fresh shoot weight of tomato was significantly
increased in T6 (Tomato plant alone, 91.35 g) as compared with T2 (solarized soil +
neem cake+ F.o ,71.75 g), T3 (solarized soil + carbendazim+ F.o, 60.95 g), T4
(solarized soil + neem cake + carbendazim+ F.o, 52.70 g) , T5 (carbendazim +
F.o,50.40 g) , T0 ( F.o alone ,7.50 g) and T1 (Solarized soil + F.o ,8.65 g) .However
among the treatments (T5,T4,T3,T2) and (T1, T0) were found non-significant from each
other (Table 4.1 and Fig. 4.2) .
Dry shoot weight (g), at 161 DAT

The results of Table 4.1 and Fig. 4.2 revealed that at 161 days after germination
the dry shoot weight of tomato was significantly increased in T6 (Tomato plant alone

,61.6 g) as compared with other treatments T2 (solarized soil + neem cake+ F.o ,39.65
g), T3(solarized soil + carbendazim+ F.o, 34.60 g), T4(solarized soil + neem cake +
carbendazim+ F.o ,30.75 g), T5 (carbendazim+ F.o, 30.45 g) , T0 ( F.o alone ,5.05 g)
and T1 (Solarized soil + F.o ,5.60 g).However (T2, T3 , T4, T5) and (T1, T0) were found
non-significant among each other.
Table 4.1: Effect of solarized soil with Neem cake and carbendazim on growth
parameters of tomato plants at different days of intervals.
Mean of the five replications
Days fresh dry
shoot length (cm) shoot shoot
weight weight
(g) (g)
Treatments 30 60 90 120 150 150 161
DAT DAT DAT DAT DAT DAT DAT
T0
F.o alone 9.70 d 24.00 c 29.65 d 40.70 c 50.65 c 7.50 c 5.05 c
T1
S.S +F.o 10.65 d 28.10 c 31.30d 44.87 c 55.10 c 8.65 c 5.60 c
T2 25.15 a 67.25 a 95.00bc 100.0ab 110.20ab 71.75ab 39.65 b
S.S + N.C + F.o
T3 20.15bc 70.00 a 100.0ab 105.25ab 114.60ab 60.95ab 34.60 b
S.S + C +F.o
T4 S.S + N.C + C 25.55 a 72.70 a 94.00bc 95.05 b 101.80 b 52.70b 30.75 b

+F.o
T5 C +F.o 20.97 b 65.75 a 85.50 c 93.65 b 100.55 b 50.40 b 30.45 b
T6 Tomato plant 16.60 c 53.15 b 108.00 a 113.05 a 124.81 a 91.35a 61.6 a

alone
18.40 54.42 62.18 84.65 93.95 161.46 118.69
Overal Mean
S S S S S S S
F- test
1.736 3.886 5.407 6.866 8.905 15.899 9.095
S. Ed. (±)
3.697 8.277 11.516 14.626 18.968 33.864 19.373
C. D. (P = 0.05)
Solarized soil = S.S
Fusarium oxysporum = F.o
Neem cake = N.C
Carbendazim = C

30 DAT 60 DAT 90 DAT 120 DAT 150 Dat
140
120
100
Shoot length (cm)
80
60
40
20
Fig. 4.1: Effect of solarized soil with Neem cake and carbendazim on shoot length
(cm) of tomato plants at different days of intervals.
T0- Fusarium oxysporum alone
T1- Solarized soil + Fusarium oxysporum
T2 - Solarized soil + neem cake+ Fusarium oxysporum
T3- Solarized soil + carbendazim +Fusarium oxysporum
T4- Solarized soil + neem cake +carbendazim + Fusarium oxysporum
T5- Carbendazim + Fusarium oxysporum
T6- Tomato plant alone

100
Fresh shoot weight Dry shoot weight
90
Fresh and dry shoot weight (g) 80
70
60
50
40
30
20
10
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Fig. 4.2: Effect of solarized soil with Neem cake and carbendazim on the fresh and
dry shoot weight (g) of tomato plants at 150 and 161 DAT.

Root length (cm) at 150 DAT
At 150 days after germination the root length of tomato in (Table 4.2 and Fig.
4.3) revealed that in T6 (Tomato plant alone ), T2 (solarized soil + neem cake+ F.o) and
T3 (solarized soil + carbendazim+ F.o) significantly increased as compared with T4
(solarized soil + neem cake + carbendazim+ F.o), T5 (carbendazim+ F.o), T1 (Solarized
soil + F.o) and T0 ( F.o alone),However (T6, T2 , T3) , (T3 , T4) , (T4 , T5) and (T1, T0)
were found non-significant among each other.
Fresh and dry root weight (g) at 150 and 161 DAT
The result of Table 4.2 and Fig. 4.4 indicates that all the treatments of solarized
soil and amended with neem cake or carbendazim significantly increased the fresh root
weight of tomato as compared with T1 (Solarized soil + F.o) and T0 (F.o alone)
Whereas the treatments (T6, T3, T2) and (T1, T0) were found non-significant among
themselves, the dry root weight of tomato in all the treatments was not significant to
each other.
Yield /plant (g) at 150 DAT

Fruit yield (g) per plant of tomato were taken of 150 days after germination in the
Table 4.2 and Fig 4.5 revealed that significantly increased the yield (g) of tomato plants
was found in T6 (170.15 g, plant without fusarium and treatments), as compared with
the treatments T3( 162.52 g ,S.S + C +F.o), T2 (145.85 g ,S.S + N.C + F.o),T4 (127.82
g, S.S + N.C + C +F.o), T5 (115.70 g ,C +F.o) , T1(0.0 g ,S.S +F.o ) and T0 (0.0 g ,F.o
alone), however the treatments (T6,T3 ) and (T2, T4) and (T4, T5) and (T1,T0 ) were found
non-significant among themselves.

Table 4.2: Effect of solarized soil with Neem cake and carbendazim on growth
parameters at 150 and 161 DAT.
Mean of the five replications

Days
root length fresh root dry root

(cm) weight (g) weight (g) Yield / plant
Treatments (g)
150 DAT 150 DAT 161 DAT 150 DAT
T0 F.o alone 10.35 d 0.60 c 0.39 a 0.00 d
T1 S.S +F.o 11.55 d 0.79 c 0.52 a 0.00 d
T2 S.S + N.C + Fro 28.60 a 4.25ab 2.60 a 145.85 ab
T3 S.S + C +F.o 27.40 ab 4.75ab 3.30 a 162.52 a
T4 S.S + N.C + C +F.o 22.65bc 3.70b 2.15a 127.82bc
T5 C +F.o 20.00 c 4.4ab 1.85a 115.70c
T6 Tomato plant alone 28.65 a 6.2a 3.30a 170.15 a
Overal Mean 21.31 14.09 2.01 103.15
F- test S S NS S
2.648 1.025 13.147

S. Ed. (±)
5.639 2.182 28.000
C. D. (P = 0.05)

Neem cake = N.C
Carbendazim = C

Root length (cm)
35
30
25
Root length (cm)
20
15
10
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Fig.4.3: Effect of solarized soil with Neem cake and carbendazim on root length
(cm) of tomato plants at 150 DAT.

7 Fresh root weight Dry root weight
6
Fresh and dry root weight (g)
5
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Fig. 4.4: Effect of solarized soil with Neem cake and carbendazim on fresh and dry
root weight (g) of tomato plants at 150 and 161 DAT.

180
160
140
Yield / plant (g)
120
100
80
60
40
20
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Fig. 4.5: Effect of solarized soil with Neem cake and carbendazim on yield / plant
(g) of tomato plants at 150 DAT.

Similar findings have been reported by El-Khallal (2007) who found marked
inhibition in the growth rate of shoot and root of tomato seedlings in response to
Fusarium wilt disease. Haruna et al. (2012) reported that application of neem leaf
based compost extract induced better plant height as compared to those that were treated
with fungicide and those in the control treatments. At 5,6,7 and 8 weeks after
transplanting tomatoes whose roots were drenched with neem-leaf based compost
(NLBC) significantly produced higher plant height. Kimaru et al. (2004); Abed et al.
(2013) reported that soil when amended with neem kernel cake powder significantly
increased stem diameter, shoot length, root length, root and shoot dry weight and
number of fruits and weight of tomato. Kumar et al. (2010) reported that integrated
treatments with neem products and Bacillus subtilis gave maximum shoot length of
tomato plants. Our findings are in agreement with Hadian et al. (2011) who reported
that application of neem seed powder to soil inoculated with either Fusarium or
Meloidogyne or both of pathogens significantly increased tomato fresh shoots weight
compared with inoculated and uninoculated pots that were not treated with neem.
Barakat and Mohammad (2012) reported that the population of Fusarium oxysporum
f.sp. lycopersici in the soil was reduced significantly after seven weeks of soil
solarization and there was increase of up to 94% in fresh plant weight and up to 60% in
plant dry weight .
The probable reasons for such findings may be that the reduction of shoot and
root length in infected seedlings could be a morphological expression of hormonal
imbalance. This hormonal imbalance in tomato seedling after Fusarium infection
induced accumulation of abscisic acid (ABA) and reduction of Auxins (IAA),
Gibberellin (GA3) and cytokinins. Steijl et al. (1999) also reported that the short root
and shoot lengths in tomato seedlings infected with Fusarium oxysporum could be due
to production of some toxins by fungi and the accumulation of phenolic compounds
produced fom the cell wall degradation mainly lignin via depolymerization results from
fungal elicitors. Farag Hanaa et al. (2011) reported that the promotive effect of neem
aqueous extract on tomato seedlings growth could be due to triterpene (azadirachtin)
which acts by delaying the transformation of ammonium nitrogen into nitrate nitrogen.
Akhtar (1999a; 1999b) observed the slow nitrogen conversion led to continuous
availability of nitrogen during plant growth and growth promotion.

4.2. Effect of solarized soil with Neem cake and carbendazim on
Disease intensity (%) and reduction (%) of Fusarium oxysporum f.sp.
lycopersici on tomato plants
At 60 days ,the disease intensity (%) of tomato plants recorded with different
treatment is presented in Table 4.3 and graphically in Figure 4.6.It was revealed from
the data that the treatments observed in T3 (solarized soil + carbendazim+ F.o), T4
(solarized soil +neem cake + carbendazim+ F.o),T5 (carbendazim+ F.o) and T2
(solarized soil + neem cake+ F.o) were found non-significant among themselves but
significantly reduced disease intensity (%) of tomato plants as compared with T1
(Solarized soil + F.o) and T0 ( F.o alone) . The reduction percentage over control are in
order T1 (15.7) smaller than T2 (68.4) smaller than T5 (73.6) smaller than T4 (78.9)
smaller than T3 (78.9) in Fig 4.7.
Results in (Table 4.3 and Fig 4.6) at 90,120 and 150 days revealed that disease
intensity (%) was significantly reduced in T3 (solarized soil + carbendazim+ F.o), T4
(solarized soil + neem cake + carbendazim+ F.o), T5 (carbendazim+ F.o), T2 (solarized
soil + neem cake+ F.o) were found non-significant among themselves but significantly
reduced disease intensity (%) of tomato plants as compared with T1 (Solarized soil +
F.o) and T0 (F.o alone), the disease intensity (%) was significantly reduced in T1 as
compared with T0. The reduction percentage is maximum in T3 followed by T4, T5, T2
and T1 as compared with T0 in Fig 4.7.

Table 4.3: Effect of solarized soil with Neem cake and Carbendazim on the disease
intensity (%) and reduction (%) of Fusarium oxysporum f.sp. lycopersici on tomato
plants
60 DAT 90 DAT 120 DAT 150 DAT
Disease Reduction Disease Reduction Disease Reduction Disease Reduction
Treatments
intensity (%) over intensity (%) over intensity (%) over intensity (%) over
(%) control (%) control (%) control (%) control
T0 F. o alone 23.7 a 0 36.2 a 0 49.9 a 0 65.3a 0
T1 S.S +F. o 20.0 a 15.7 25.0 b 31.0 30.8b 38.1 41.4b 36.6
S.S + N.C
T2 7.5 b 68.4 15.0 c 58.6 20.0c 59.9 21.5c 67.1
+F. o
T3 S.S + C +F. o 5.0 b 78.9 11.2 c 68.9 15.0c 69.9 16.2 c 75.1
S.S + N.C + C
T4 5.0 b 78.9 12.5 c 65.5 15.0c 69.9 16.7 c 74.3
+F. o
T5 C +F. o 6.2 b 73.6 12.4 c 65.5 16.2c 67.4 17.2 c 73.6
Tomato plant
T6 0.0 b 100.0 0.0 d 100.0 0.0d 100.0 0.0d 100.0
alone
Overal Mean 9.64 17.42 23.71 27.88
F- test S S S S
S. Ed. (±) 4.725 4.481 3.549 3.154
C. D.(P =0.05) 10.063 9.545 7.559 6.719

Neem cake = N.C
Carbendazim = C

70 60 DAT 90 DAT 120 DAT 150 DAT
60
50
Disease intensity (%)
40
30
20
10
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Fig.4.6: Effect of solarized soil with Neem cake and carbendazim on the disease
intensity (%) of Fusarium oxysporum f.sp. lycopersici on tomato plants at different
days of intervals.

120 60 DAT 90 DAT 120 DAT 150 DAT
100
80
Reduction (%)
60
40
20
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Fig.4.7: Effect of solarized soil with Neem cake and carbendazim on the reduction
(%) of Fusarium oxysporum f.sp. lycopersici on tomato plants at different days of
intervals.

Similar findings have been reported by Farag Hanaa et al. (2011) who
concluded that neem aqueous extracts pretreatments prevented the growth inhibition
caused by Fusarium oxysporum. Kumar et al. (2010) reported that integrated
treatments with neem products and Bacillus subtilis gave maximum disease control
(65.33%) of tomato.Hadian et al. (2011) reported that Neem seed powder significantly
(P # 0.01) reduced the disease severity of Fusarium of tomato plants .
Abed et al. (2013) reported that the carbendazim was the best treatment with
lowest disease severity of Fusarium oxysporum f.sp. lycopersici wilt of tomato during
30,45,60,75 days after transplanting .Whereas, maximum disease severity was recorded
in control pot. All treatments were significantly reduced the disease severity compared
to control.
The probable reasons for such findings may be that Neem plant possesses
biologically active compounds mainly alkaloids such as isoprenoids that control various
pest and diseases of Fusarium oxysporum and Meloidogyne incognita complex of
tomato (Hadian et al. 2011). Sadre et al. (1983) reported the significant reduction of
fusarium wilt disease in tomato plants treated with neem extract could be due to the
presence of gedunin i.e. tetranortriterpenoid which posses antifungal properties or due
to presence of Azadirachtin (tetranortriterpenoid).Oliveira(1992) reported that counts of
populations from naturally infested soil samples indicated that both pathogenic and
nonpathogenic Fusariurn spp were markedly reduced by solarization at the upper 20 cm
soil layer.

60 day
T0
T1
150 days
T0 T1
Plate 4.1: Pathogenicity test of F. oxysporum on tomato plants with solarized soil
(T1) Healthy plant (T0) Infected plant

60 day
T1 T2 T4
150 days
T1 T2 T4
Plate 4.2: (T1) solarized soil + F.o, (T2) Neem cake + F.o and (T4) Neem cake +
carbendazim + F.o

60 day
T1 T3 T5
150 days
T1 T3 T5
Plate 4.3: (T1) solarized soil + F.o, (T3) solarized soil + carbendazim+ F.o and (T5)
Carbendazim + F.o

60 day
T0 T1 T2 T3 T4 T5 T6
150 days
T0 T1 T2 T3 T4 T5 T6
Plate 4.4: (T0) F.o alone, (T1) solarized soil + F.o (T2) Neem cake + F.o, (T3)
solarized soil +carbendazim + F.o, (T4) Neem cake + carbendazim + F.o, (T5)
Carbendazim + F.o and (T6) tomato plant alone

4.3. Effect of carbendazim and solarized soil with spent mushroom
compost, Pseudomonas fluorescens on plant growth parameters of
tomato plants
The results of Table 4.4 and Fig. 4.8 revealed the significant increase in the shoot
length of tomato in the treatments T5 (carbendazim + F.o ,20.97 cm) as compared with
other treatments including T6 (tomato plant alone ,16.60 cm) ,T4 (solarized soil + spent
mushroom compost + Pseudomonas fluorescens , 17.17 cm) . Among the treatments,T3
(solarized soil + Pseudomonas fluorescens , 14.45 cm) significantly increased the shoot
length from T0 (F.o alone, 9.70 cm), T1 (solarized soil + F.o ,10.65 cm) ,T2 (solarized
soil + spent mushroom compost, 13.25 cm), However (T4, T6 , T3) , and (T2, T1,T0)
were found to be non-significant from each other respectively.
The shoot length of tomato were significantly increased in the treatments of

T5(carbendazim + F.o ,65.75 cm) and T4 (solarized soil + Pseudomonas fluorescens +
spent mushroom compost ,58.05 cm) as compared with T6 (Tomato plant alone, 53.15
cm),T2 (solarized soil + spent mushroom compost ,45.75 cm) and T3 (solarized soil +
Pseudomonas fluorescens,41.00cm).The treatments of spent mushroom compost alone
or in combination with Pseudomonas fluorescens significantly increased the shoot
length as compared to the treatments including T0 ( F.o alone, 24.00 cm) and T1 (
solarized soil + F.o , 28.10 cm ) However (T5, T4) , (T4, T6) (T6, T2) , (T2, T3) and (T0 ,
T1) were found non-significant from each other in (Table 4.4 and Fig. 4.8).
The results of Table 4.4 and graphically represented in Fig. 4.8 indicated that
there were significantly increase in the shoot length (cm) in T6 (Tomato plant
alone,108.00 cm) from other treatments .Among the treatments T4 (solarized soil +
spent mushroom compost + Pseudomonas fluorescens ) and T3 (solarized soil +
Pseudomonas fluorescens) ;T0 (F.o alone ) and T1 ( solarized soil + F.o ) were found
non-significant from each other.

The results of Table 4.4 and Fig. 4.8 revealed that the shoot length was found
significantly increase in T6 (Tomato plant alone, 113.05 cm) from other treatments.The
treatments T4 (solarized soil + spent mushroom compost + Pseudomonas
fluorescens,98.10 cm) and T5(carbendazim + F.o ,93.65 cm) significantly increased
from T2 (solarized soil + spent mushroom compost ,78.25 cm), T1 ( solarized soil + F.o
) and T0 ( F.o alone ). Among the treatments (T3, T2) and (T1, T0) were found non-
significant from each other.
The results of Table 4.4 and graphically represented in Fig. 4.8 indicated that the
treatments T6 (Tomato plant alone) and T4 (solarized soil + spent mushroom compost
+Pseudomonas fluorescens) significantly increased the shoot length (cm) from T3
(solarized soil + Pseudomonas fluorescens , 95.50 cm) and T2 (solarized soil + spent
mushroom compost, 81.25 cm) , T1 ( solarized soil + F.o ,55.10 cm) and T0 ( F.o
alone , 50.65 cm).Among the treatments (T6, T4), (T4, T5), (T5, T3), (T3, T2) and (T1, T0)
were found non-significant from each other.
Fresh and dry shoot weight (g) at 150 and 161DAT

The results of Table 4.4 and Fig. 4.9 revealed the significant increase of fresh and
dry shoot weight (g) of tomato plants was found in T6 (Tomato plant alone ) as
compared with other treatments. Among the treatments T5 (carbendazim + F.o), T4
(solarized soil + spent mushroom compost + Pseudomonas fluorescens), T3 (solarized
soil + Pseudomonas fluorescens) and T2 (solarized soil + spent mushroom compost)
significantly increased of fresh and dry shoot weight (g) from T1 (solarized soil + F.o)
and T0 (F.o alone). However, (T0, T1); (T2, T3, T4, T5) were non-significant from each
other.

Table 4.4: Effect of carbendazim and solarized soil with spent mushroom compost,
Pseudomonas fluorescens on growth parameters of tomato plants at different days
of intervals.
Mean of five replication
Days fresh dry
weight weight
(g) (g)
Treatments 30 60 90 120 150 150 161
T0 9.70 d 24.00 e 29.65d 40.70 d 50.65 e 7.50 c 5.05 c

F.o alone
T1 10.65 d 28.10 e 31.30 d 44.87 d 55.10 e 8.65 c 5.60 c

S.S +Fo
13.25cd 45.75 cd 78.00 c 78.25 c 81.25 d 40.7 b 21.95 b
T2 S.S + S.m.c +
F.o
T3 14.45 bc 41.00 d 86.00 bc 89.35 bc 95.50 cd 37.20 b 22.80 b
S.S +P. f +F.o
T4 S.S +S.m.c+P. f 17.17 b 58.05 ab 96.00 b 98.10 b 112.00 ab 62.20 b 36.40b

+Fro
T5 20.97 a 65.75 a 85.50 c 93.65 b 100.55 bc 50.40 b 30.45 b

C +F.o
T6 Tomato plant 16.60 bc 53.15 bc 108.00 a 113.05 a 124.81 a 91.35 a 61.60 a

alone
14.69 45.11 58.06 79.71 88.54 179.60 105.06
Overal Mean
S S S S S S S
F- test
1.688 4.498 5.149 5.808 7.146 12.348 8.099
S. Ed. (±)
3.595 9.580 10.966 12.372 15.221 26.302 17.251
C. D. (P = 0.05)
Spent mushroom compost = S.m.c
Carbendazim = C
Pseudomonas fluorescens=P. f

30 DAT 60 DAT 90 DAT 120 DAT 150 DAT
140
120
100
Shoot length (cm)
80
60
40
20
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Fig. 4.8: Effect of carbendazim and solarized soil with spent mushroom compost,
Pseudomonas fluorescens on the shoot length (cm) of tomato plants at different
days of intervals.
T2 - Solarized soil + spent mashroom compost+ Fusarium oxysporum
T3- Solarized soil + Pseudomonas fluorenscens +Fusarium oxysporum
T4- Solarized soil + spent mashroom compost +Pseudomonas fluorenscens +

Fusarium oxysporum

100 Fresh shoot weight Dry shoot weight
90
Fresh and dry shoot weight (g) 80
70
60
50
40
30
20
10
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Pseudomonas fluorescens on the fresh and dry shoot weight (g) of tomato plants at
150 and 161 days.
T2 - Solarized soil + spent mashroom compost + Fusarium oxysporum

Fusarium oxysporum

Root length (cm) of at 150 DAT
Results of Table 4.5 and Fig. 4.10 observed that T6 (Tomato plant alone)
significantly increased the root length (cm) of tomato from other treatments T4
(solarized soil + spent mushroom compost + Pseudomonas fluorescens), T3 (solarized
soil + Pseudomonas fluorescens) and T2 (solarized soil + spent mushroom compost)
significantly increased from T5 (carbendazim + F.o), T1 (solarized soil + F.o) and T0
(F.o alone).
Fresh and dry root weight (g) at 150 and 161 DAT
The result of Table 4.5 and Fig. 4.11 indicates that significantly increased the
fresh root weight in T6 (Tomato plant alone, 6.2 g) from other treatments. T5
(carbendazim + F.o ,4.40 g) ,T2 (solarized soil + spent mushroom compost ,4.35 g) and
T4 (solarized soil + spent mushroom compost + Pseudomonas fluorescens ,3.80 g) were
found non-significant from each other but they are significantly increased from T3
(solarized soil + Pseudomonas fluorescens ,2.60 g ) , T1 ( solarized soil + F.o , 0.79 g)
and T0 ( F.o alone,0.60 g) , the dry root weight of tomato in all the treatments was not
significant to each other.
Yield / plant (g) at 150 DAT

Table 4.5 and Fig 4.12 revealed that significantly increased the yield (g) of tomato
plants was found in T6 (170.15 g, plant without fusarium and treatments), as compared
with the treatments T2( 128.25 g, S.S + S.m.c + F.o),T3( 121.80 g ,S.S +P. f +F.o), T5
(115.70 g ,C +F.o) ,T4 (106.75 g ,S.S + S.m.c +P. f +F.o) , T1(0.0 g ,S.S +F.o ) and T0
(0.0 g ,F.o alone), however the treatments (T2,T3,T5) and (T3,T5,T4) and (T1,T0 ) were
found non-significant among themselves.

Pseudomonas fluorescens on growth parameters at 150 and 161 DAT.
Days Mean of five replication
root length fresh root dry root Yield /

(cm) weight (g) weight (g) plant (g)
Treatments
150 DAT 150 DAT 161 DAT 150 DAT
T0 F.o alone 10.35 d 0.60 d 0.39 a 0.00 d
T1 S.S +Fro 11.55 d 0.79 d 0.52 a 0.00 d
T2 23.85 bc 4.35 b 1.90 a 128.25 b

S.S + S.m.c + F.o
T3 25.70 ab 2. 60 c 1.10 a 121.80 bc
S.S +P. f +F.o
T4 26.95 ab 3. 80 b 1. 40 a 106.75 c
S.S +S.m.c+P. f +F.o
T5 C +Fro 20.00 c 4.40 b 1.85 a 115.77 bc
T6 Tomato plant alone 28.65 a 6. 20 a 3.30 a 170.15 a
Overal Mean 21.01 12.97 5.98 91.82
F- test S S NS S
2.066 0.518 9.641

S. Ed. (±)
C. D. (P = 0.05) 4.401 1.103 20.536

Spent mashroom compost = S.m.c
Carbendazim = C
Pseudomonas fluorenscens =P. f

35 Root length (cm)
30
25
Root length (cm)
20
15
10
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Fig.4.10: Effect of carbendazim and solarized soil with spent mushroom compost,
Pseudomonas fluorescens on the root length (cm) of tomato plants at 150 DAT.

Fusarium oxysporum

7 Fresh root weight Dry root weight
6
5
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Pseudomonas fluorescens on the fresh and dry root weight (g) of tomato plants at
150 and 161 DAT.

Fusarium oxysporum

180
160
140
Yield / plant (g)
120
100
80
60
40
20
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Pseudomonas fluorescens on the yield / plant (g) of tomato plants at 150 DAT.

Fusarium oxysporum

Similar findings have been reported by Borrero et al. (2009) observed that the
treatment Spent mushroom compost mixed with peat was recorded minimum shoot
length of carnation plants with no significant differences from other treatments.
Saravanan et al. (2013) reported that inoculation with Pseudomonas fulorescens Pf5
induced a significant increase in shoot length, root length and dry weight of tomato
plants. Treatment of plants with either F. oxysporum or R. solani drastically reduced the
shoot length, root length and dry weight of the plant. However, in the presence of
Pseudomonas fulorescens the adverse effect of the pathogens on growth of tomato
plants was alleviated. Mehnaz and Lazarovits (2006) reported an increase in root and
shoot weight of corn plants upon inoculation with a strain of Pseudomonas putida that
also possess antagonistic activity against Fusarium. Meenakshisundaram and
Santhagur (2012) indicate that shoot biomass was slightly greater in inoculated plants
with Pseudomonas fluorenscens than in control plants. Akköprü and Demir (2005)
reported the effects of the arbuscular mycorrhizal fungus (AMF) Glomus
intraradices and four rhizobacteria (RB; 58/1 and D/2: Pseudomonas fluorescens
biovar II; 17: P. putida; 21:Enterobacter cloacae), which are the important members of
the rhizosphere microflora and biological control agents against plant diseases, were
examined in the pathosystem of Fusarium oxysporum f.sp. lycopersici (FOL) and
tomato with respect to morphological parameters (fresh and dry root weight). Individual
bacteria inoculations were more effective than both the single AMF and dual (Glomus
intraradices + RB) inoculations. In addition, the RB and Glomus intraradices enhanced
dry root weight effectively. Significant increases in root weights were recorded
particularly in the triple inoculations compared with single or dual inoculations.
The probable reasons for such findings may be that suitable combinations of these
biocontrol agents may ameliorate plant growth and health. Pseudomonas spp promote
plant growth by secreting auxins, gibberellins and cytokinins (Thangavelu et al., 2003;
Haas and Défago 2005; Gravel et al., 2007; Yao et al., 2010). Martyn and Hartz
(1986) reported that Soil solarization for either 30 or 60 days was effective in delaying
the onset of wilt symptoms as well as in reducing total disease incidence in a Fusarium-
susceptible watermelon cultivar, Sugarbaby but complete disease control was not
achieved.

compost, Pseudomonas fluorescens on Disease intensity (%) and
reduction (%) of Fusarium oxysporum f.sp. lycopersici on tomato plants
At 60 days, the disease intensity (%) of tomato plants recorded with different
treatments is presented in Table 4.6 and graphically in Figure 4.13. It was revealed from
the data that the treatments T5 (carbendazim + F.o, 6.25 %), T4 (solarized soil +
Pseudomonas fluorescens + spent mushroom compost, 7.50 %), T3 (solarized soil +
Pseudomonas fluorescens, 8.75%), and T2 (solarized soil + spent mushroom compost,
10.00 %) are significantly reduced the incidence of Fusarium oxysporum as compared
with T1, T0. Among the treatments non significant disease intensity (%) was found in T0
(F.o alone, 23.75 %) and T1 (solarized soil + F.o, 20.00 %). The reduction percentage
over control are in order T1 (15.7) smaller than T2 (57.8) smaller than T3 (63.1) smaller
than T4 (68.4) smaller than T5 (73.6) in Fig 4.14.
Observation recorded on 90,120,150 days as shown in Table 4.6 and Fig 4.13
pertaining to mean disease intensity (%) of tomato plants reveal that significantly
reduced in T5 (12.4), T4 (15.0), T3 (16.2) and T2 (21.2%) as compared toT0 (36.2%) at 90
days after germination, the treatments (T2, T3, T4, T5) are not significant each other, the
reduction percentage is maximum in T5 followed by T4, T3, T2 and T1 as compared to T0
in Fig 4.12. At 120 and 150 days of germination the treatments significantly reduced the
disease intensity in T5 (16.2%) and (17.2%) as compared with T0 (49.9%) and (65.3%)
respectively , the treatments (T5, T4, T3 and T2) were found not significant from each
other as compared with T0 and T1. disease intensity reduction % F. oxysporum f. sp.
Lycopersici was significantly reduced in T5, T4, T3, and T2 at 120 days of germination
from T1 (30.8 %) ,T0 (49.9 %) and at 150 days of germination from T1 (41.4 %) ,T0
(65.3 %).

Table 4.6: Effect of Carbendazim and solarized soil with Pseudomonas fluorenscens,
spent mushroom compost on the disease intensity (%) and reduction (%) of Fusarium
oxysporum f.sp. lycopersici on tomato plants
Treatment
T0 F. o alone 23.7a 0 36.2 a 0 49.9 a 0 65.3 a 0
T1 S.S +F. o 20.0ab 15.7 25.0 ab 31.0 30.8b 38.1 41.4b 36.6
S.S + S.m.c +
T2 10.0bc 57.8 21.2bc 41.3 25.0bc 49.8 26.0c 60.2
F. o
T3 S.S + Pf +F. o 8.7bc 63.1 16.2bc 55.1 20.0 c 59.9 23.0c 64.7
S.S + S.m.c +
T4 7.5bc 68.4 15.0bc 58.6 18.7c 62.4 21.2c 67.4
P.f +F. o
T5 C +F. o 6.2c 73.6 12.4c 65.5 16.2c 67.4 17.2c 73.6

Tomato plant
T6 0.0c 100 0.0d 100 0.0d 100 0.0d 100
alone
Overal Mean 10.89 19.46 24.96 30.14
F- test S S S S
S. Ed. (±) 6.013 5.261 4.615 5.065
C. D.(P =0.05) 12.80 11.20 9.83 10.78

Carbendazim = C
Pseudomonas fluorenscens=P. f

70
60
50
Disease intinsity (%)
40
30
20
10
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Pseudomonas fluorescens on the disease intensity (%) of Fusarium oxysporum f.sp.
lycopersici on tomato plants at different days of intervals.
T2 - Solarized soil + spent mushroom compost + Fusarium oxysporum
T4- Solarized soil + spent mushroom compost +Pseudomonas fluorenscens +

Fusarium oxysporum

120 60 DAT 90 DAT 120 DAT 150 DAT
100
80
Reduction (%)
60
40
20
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Pseudomonas fluorescens on the Reduction (%) of Fusarium oxysporum f.sp.
lycopersici on tomato plants at 60, 90, 120, 150 days.

Fusarium oxysporum

Similar findings have been reported by Rocha and Andrea (2013) reported that
microbiolization of seeds with Pseudomonas sp reduced the tomato wilt in both assays
(36.6 and 91.7% in the first and second assays respectively. Seleim et al. (2011)
indicated that Pseudomonas fluorescens exhibited the highest disease reduction
percentage of tomato bacterial wilt disease (80%) followed by Pseudomonas putida and
then Bacillus subtilis. Kamilova et al. (2006) reported that P. fluorescens reduced the
disease pressure caused by F. oxysporum f. sp. radicis-lycopersici from 96 to 7%.
Borrero et al. (2009) reported that the suppressiveness of these composts was extended
to another formae speciales (F. oxysporum f.sp. dianthi). For both Fusarium wilts, grape
marc compost (GMC) was the most suppressive.The carnation Fusarium wilt
suppressiveness registered for olive oil husk + cotton gin trash composted and mixed
with rice husk (OC+R) and Spent mushroom compost mixed with amended light peat
(SM + P) was also established for tomato Fusarium wilt (Borrero et al. 2005).
Rajput et al. (2006) reported that the most effective fungicides in reducing the
growth of F.oxysporum f.sp. vasinfectum as compared to control was Carbendazim
followed by Thiophanate-methyl.
The probable reasons for such findings may be that production of siderophores
and chitinases are two factors that may be involved Pseudomonas fluorenscens
biological control activity (Meenakshisundaram and Santhaguru 2012). It is known
that chitinolytic activity and siderophore production are correlated with antifungal
activity (Castoria et al., 2001; Kamensky et al., 2003; Quecine et al., 2008). In
addition, Pseudomonas fluorenscens is capable of solubilizing phosphate and producing
IAA, characteristics that may enhance its potential use as an effective biological control
agent to contribute to the control of tomato wilt-off caused by F. oxysporum (Ardebili
et al., 2011). In addition to promoting plant growth, Pseudomonas fluorescens Pf1 as an
effective biocontrol agent reduced several plant diseases (Manikandan et al., 2010).
Mechanisms of biological control by which rhizobacteria can promote plant growth
indirectly, i.e., by reducing the level of disease, include antibiosis, induction of systemic
resistance, competition for nutrients and niches (Lugtenberg and Kamilova 2009).
Biocontrol pseudomonas implicated in the suppression of fungal root diseases by
various mechanisms including production of antibiotics,toxins, bio-surfactants, or lytic
enzymes, competition forcolonization sites, nutrients and minerals, and induction of
systemic resistance (Erdogan and Benlioglu 2010). The production of antifungal
metabolites proposed asa main mechanism of antifungal activity of Pseudomonas

fluorescens against Fusarium oxysporum f.sp. lycopersici (Kumar et al.2002;
Karkachi et al. 2010). P. fluorescensWCS365 efficiently suppressed Fusarium
oxysporum (Woeng et al. 1998; Dekkers et al. 2000; Bolwerk et al. 2003).
Ramamoorthy et al. (2002) reported that P. fluorescens isolate Pf1 increased plant
vigour and consistently reduced the disease incidence under greenhouse condition sand
disease protection was comparable with fungicide, carbendazim. Similarly, under field
conditions, the talc-based bio-formulation containing P. fluorescens isolate Pf1 was
found to protect the plants against wilt.

60 day
T1 T3 T4
150 days
T1 T3 T4
Plate 4.5: (T1) solarized soil + F.o, (T3) Pseudomonas fluorescens + F.o and (T4)
spent mushroom compost + Pseudomonas fluorescens + F.o

60 day
T1 T3 T5
150 days
T1 T3 T5
Plate 4.6: (T1) solarized soil + F.o, (T3) Pseudomonas fluorescens + F.o and (T5)
carbendazim + F.o

60 day
T1 T2 T4 T5
150 days
T1 T2 T4 T5
Plate 4.7: (T1) solarized soil + F.o, (T2) spent mushroom compost + F.o, (T4) spent
mushroom compost + Pseudomonas fluorescens + F.o and (T5) carbendazim + F.o

60 day
T0 T1 T2 T3 T4 T5 T6
150 days
T0 T1 T2 T3 T4 T5 T6
Plate 4.8: (T0) F.o alone, (T1) solarized soil + F.o (T2) spent mushroom compost +
F.o, (T3) Pseudomonas fluorescens + F.o, (T4) spent mushroom compost +
Pseudomonas fluorescens + F.o, (T5) carbendazim + F.o and (T6) tomato plant
alone

compost, Trichoderma harzianum on plant growth parameters of
tomato plants

The results of Table 4.7 and Fig. 4.15 revealed that the shoot length of tomato in
T5(carbendazim+ F.o) significantly increased from T6 (Tomato plant alone) ,
T3(solarized soil + Trichoderma harzianum+ F.o) , T2 (solarized soil + Spent
mushroom compost+ F.o) , T1 ( solarized soil + F.o) and T0 (F.o alone), However (T5,
T4) , (T4,T6,T3) ,(T6,T3,T2) and (T2,T1, T0) ,were found non-significant among
themselves.

At 60 days after germination of tomato plants the treatment T5 (carbendazim+
F.o, 65.75 cm) significantly increased the shoot length from other treatments including
T0 (F.o alone). Whereas the treatments (T0, T1), (T2, T3, T4, T6) were found non-
significant from each other in (Table 4.7and Fig. 4.15).

The results of Table 4.7 and graphically represented in Fig. 4.15 indicated that
there were significantly increased in the shoot length (cm) in the treatments T6 (Tomato
plant alone ,108.00 cm),as compared with T5(carbendazim+ F.o, 85.50 cm), T2
(solarized soil + Spent mushroom compost+ F.o, 78.00 cm) , T1 (solarized soil +
F.o,31.30 cm) and T0 (F.o alone ,29.65 cm). Among the treatments (T6, T4, T3), (T3,
T5), (T5, T2) and (T0, T1) were found non-significant from each other.
Shoot length (cm), at 120 and 150 DAT

The results of Table 4.7 and Fig. 4.15 revealed the significant increase of shoot
length in T6 (Tomato plant alone) as compared with T5 (carbendazim+ F.o ) ,T3
(solarized soil + Trichoderma harzianum+ F.o) , T2 (solarized soil + Spent mushroom
compost+ F.o) ,T1 ( solarized soil + F.o) and T0 (F.o alone), However (T6, T4) , (T3, T4)
,(T3, T5) and (T1, T0) ,were found non-significant from each other.

Fresh and dry shoot weight (g) at 150 and 161 DAT
The fresh and dry shoot weight (g) of tomato plants recorded with different
treatment is presented in Table 4.7 and graphically in Figure 4.16. It was revealed from
the data that the treatments of T6 (Tomato plant alone) significantly increased fresh
shoot weight from other treatments followed by T4 (solarized soil + Spent mushroom
compost + Trichoderma harzianum+ F.o), T3 (solarized soil + Trichoderma harzianum+
F.o) and T5 (carbendazim+ F.o), However (T4, T3, T5), (T4, T5, T2) and (T1, T0), were
found non-significant from each other, the dry shoot weight of tomato in all the
treatments was not significant to each other.

Trichoderma harzianum on growth parameters of tomato plants at different days of
intervals.

Days fresh dry
weight weight
(g) (g)
Treatments 30 60 90 120 150 150 161
T0 F.o alone 9.70 d 24.00 c 29.65 d 40.70 e 50.65 e 7.5 d 5.05 a
T1 10.65 d 28.10 c 31.30 d 44.87 e 55.10 e 8.65 d 5.60 a

S.S +F.o
T2 S.S + S.m.c+ 13.25 cd 45.75 b 78.00 c 78.25 d 81.25 d 40.70 c 21.95 a

F.o
T3 S.S + T.h +F.o 14.85 bc 47.30 b 96.00 ab 96.75 b 103.45 bc 53.45 bc 32.80 a
T4 S.S + S.m.c+ 17.70 ab 49.85 b 98.00 a 106.75 ab 118.10 ab 67.25 b 42.20 a

T.h +F.o
T5 C +F.o 20.97 a 65.75 a 85.50 bc 93.65 b 100.55 c 50.40 bc 30.45 a
T6 Tomato plant 16.60 bc 53.15 b 108.0 a 113.05 a 124.81 a 91.35 a 61.60 a

alone
Overal Mean 14.82 44.84 60.04 81.86 89.27 179.60 109.00
S S S S S S NS
F- test
1.865 4.501 5.808 6.530 7.983 11.375
S. Ed. (±)
3.973 9.588 12.371 13.908 17.003 24.228
C. D. (P = 0.05)
Spent mushroom compost = S.m.c
Carbendazim = C
Trichoderma harzianum=T. h

30 DAT 60 DAT 90 DAT 120 DAT 150 DAT
140
120
100
Shoot length (cm)
80
60
40
20
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Trichoderma harzianum on the shoot length (cm) of tomato plants at different days
of intervals.
T3- Solarized soil + Trichoderma harzianum+Fusarium oxysporum
T4- Solarized soil + spent mashroom compost +Trichoderma harzianum + Fusarium

oxysporum

Fresh shoot weight Dry shoot weight
100
90
Fresh and dry shoot weight (g)
80
70
60
50
40
30
20
10
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Trichoderma harzianum on the fresh and dry shoot weight (g) of tomato plants at
150 and 161 days.

oxysporum

Root length (cm) at 150 DAT
Results of root length of tomato are presented in the Table 4.8 and Fig. 4.17
observed that T6 (Tomato plant alone,28.65 cm), T4 (solarized soil + Spent mushroom
compost +Trichoderma harzianum+ F.o ,28.35 cm), T3 (solarized soil + Trichoderma
harzianum+ F.o ,28.25 cm) significantly increased from T5 (carbendazim+ F.o ,20.00
cm) , T1 ( solarized soil + F.o ,11.55 cm) and T0 (F.o alone,10.35 cm). Among the
treatments (T0, T1), (T3, T4, T6, T2), (T5, T2) were not significant from each other.
Fresh root weight (g) at 150 DAT

The result of Table 4.8 and Fig. 4.18 indicates that significantly increased the
fresh root weight in treatment T6 (Tomato plant alone, 6.20 g) from other treatments,
including (T0, T1). Among the treatments T4 (solarized soil + spent mushroom compost
+ Trichoderma harzianum+ F.o ,4.65 g) , T3 (solarized soil + Trichoderma harzianum+
F.o ,4.50 g) ,T5 (carbendazim+ F.o,4.40 g) and T2 (solarized soil + spent mushroom
compost+ F.o ,4.35 g) were found non-significant among themselves as compared with
T1 ( solarized soil + F.o ,0.79 g) and T0 (F.o alone,0.60 g).
Dry root weight (g) at 161 DAT

The data presented in Table 4.8 and Fig. 4.18 observed that significantly
increased the dry root weight in the treatments T6 (Tomato plant alone,3.30 g) as
compared with T5 (carbendazim+ F.o,1.85 g),T2 (solarized soil + spent mushroom
compost+ F.o ,1.90 g) , T3 (solarized soil + Trichoderma harzianum+ F.o ,1.60 g) , T1
( solarized soil + F.o ,0.52 g ) and T0 (F.o alone,0.39 g) . However the treatments (T6,
T4), (T4, T3, T5, T2) and (T1, T0) were found non-significant from among themselves.
Yield / plant (g) at 150 DAT

Table 4.5 and Fig 4.12 revealed that significantly increased the yield (g) of tomato
plants was found in T6 (170.15 g, plant without fusarium and treatments), as compared
with the treatments T4 (137.52 g ,S.S + S.m.c + T.h +F.o) , T2( 128.25 g, S.S + S.m.c +
F.o), T5 (115.70 g ,C +F.o) ,T3( 107.12 g ,S.S + T.h +F.o), T1(0.0 g ,S.S +F.o ) and T0
(0.0 g ,F.o alone), however the treatments (T4, T2) and (T2,T3,T5) and (T1,T0 ) were
found non-significant among themselves.

Trichoderma harzianum on growth parameters at 150 and 161 DAT.

Days
root length fresh root dry root Yield /
Treatments plant (g)
(cm) weight (g) weight (g)
150 DAT 150 DAT 161 DAT 150 DAT
T0 F.o alone 10.35 c 0.60 c 0.39 d 0.00 d
T1 S.S +F.o 11.55 c 0.79 c 0.52 d 0.00 d
T2 S.S + S.m.c+ F.o 23.85 ab 4.35 b 1.90 b 128.25 bc
T3 S.S + T.h +F.o 28.25 a 4.50 b 1.60 b 107.12 c
S.S + S.m.c+ T.h

T4 28.35 a 4.65 b 2.55 ab 137.52 b
+F.o
T5 C +F.o 20.00 b 4.40 b 1.85 b 115.77 c
T6 Tomato plant alone 28.65 a 6.20 a 3.30 a 170.15 a
Overal Mean 21.57 14.54 6.93 94.18
F- test S S S S
2.799 0.741 0.459 8.238

S. Ed. (±)
5.961 1.579 0.977 17.546
C. D. (P = 0.05)
Carbendazim = C

Root length (cm)
35
30
25
Root length (cm)
20
15
10
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Trichoderma harzianum on the root length (cm) of tomato plants at 150 DAT.

oxysporum

Fresh root weight Dry root weight
7
6
5
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Trichoderma harzianum on the fresh and dry root weight (g) of tomato plants at
150 and 161 DAT.

oxysporum

180
160
Yield / plant (g)
140
120
100
80
60
40
20
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Trichoderma harzianum on the yield / plant (g) of tomato plants at 150 DAT.

oxysporum

Similar findings have been reported by Bokhari and Kahkashan, (2012) reported
that under pots conditions, tomato plants treated with Trichoderma harzianum showed
117.5, 138.9 % increase in shoot length and dry shoot weight respectively compared to
the control. Sundaramoorthy and Balabaskar,(2013) reported that tomato plants
treated with Trichoderma harzianum (ANR-1) isolate showed a significant stimulatory
effect on shoot length (by 73.62 cm) of tomato plants in comparison to other isolates
and untreated control. Morsy et al.(2009) revealed that the shoot length of tomato
plants were significantly increased with the Trichoderma viride. Azarmi et al. (2011)
reported that increased in shoot length, fresh and dry root weight in tomato seedlings
were observed when sown in Trichoderma sp. isolate of T.harzianum T969 made
fortified soil when compared with the control, seedling height, fresh and dry root weight
also increased in seed inoculated treatments by Trichoderma isolates.Alwathnani and
Kahkashan, (2012) reported that the plant fresh and dry weight of tomato was found to
be significantly increased in all treatments as compared to F. oxysporum f. sp.
lycopersici FOL inoculated control. Maximum fresh plant weight was shown by
Trichoderma harzianum (1.81 g). Abed et al. (2013) reported that the maximum root
length (19.00 cm) was recorded in Trichoderma harzianum, followed by (18.08 cm),
Trichoderma viride, (17.00 cm), including with control (10.2 cm).
The probable reasons for such findings may be that Trichoderma harzianum have
many positive effects on plant growth, yield, nutrient uptake, fertilizer utilization
efficiency, rate of seed germination and systemic resistance to plant diseases (Harman
et al., 2004; Harman, 2006). The increase in plant growth might be associated with
secretion of auxins, gibberellins and cytokinins (Sundaramoorthy and Balabaskar,
2013).

compost, Trichoderma harzianum on Disease intensity (%) and
reduction (%) of Fusarium oxysporum f.sp. lycopersici on tomato plants
At 60 days, the disease intensity (%) of tomato plants is presented in Table 4.9
and graphically in Figure 4.20 that significantly reduced the incidence of Fusarium
oxysporum in the treatments T5 (carbendazim+ F.o ,6.25 %) ,T4 (solarized soil + Spent
mushroom compost + Trichoderma harzianum+ F.o ,7.50 %), T3 (solarized soil +
Trichoderma harzianum+ F.o ,7.50 %) and T2 (solarized soil + Spent mushroom

compost+ F.o ,10.00 %) as compared with T0 (F.o alone, 23.75 %) . However the
treatments (T4, T3, T2), (T2, T1) and (T1, T0) were found non-significant from among
themselves. The reduction percentage over control are in order T1 (15.7) smaller than T2
(57.8) smaller than T3 and T4 (68.35) smaller than T5 (73.6) in Fig 4.21.
Observation recorded on 90,120,150 days as shown in Table 4.9 and Fig 4.20
pertaining to mean disease intensity (%) of tomato plants reveal that significantly
reduced in T5 (carbendazim+ F.o),12.50,16.25,17.25 %) ,T4 (solarized soil + Spent
mushroom compost + Trichoderma harzianum+ F.o ,13.75,16.20,19.05 %), T3
(solarized soil + Trichoderma harzianum+ F.o,15.00,17.50,20.15 %) and T2 (solarized
soil + Spent mushroom compost+ F.o ,21.25,25.00,26.00 %) were found non-
significant among themselves but significantly reduced disease intensity (%) of tomato
plants as compared with T1 ( solarized soil + F.o ,25.00,30.8,41.4 %) and T0 (F.o alone
,36.2 ,49.9,65.3 %) whereas (T1 and T0) was found significant different from each other.
The reduction percentage is maximum in T5 followed by T4, T3, T2 and T1 as compared
to T0 in Fig 4.21.

Trichoderma harzianum on the disease intensity (%) and reduction (%) of
Fusarium oxysporum f.sp. lycopersici on tomato plants

Treatment
T0 F. o alone 23.7 a 0 36.2 a 0 49.9 a 0 65.3 a 0
T1 S.S + F. o 20.0 ab 15.7 25.0 b 31.0 30.8 b 38.1 41.4 b 36.6
S.S + S.m.c
T2 10.0 bc 57.8 21.2 bc 41.3 25.0 bc 49.8 26.0 c 60.2
+F. o
T3 S.S + T.h+F. o 7.50 c 68.35 15.0 bc 58.56 17.5 c 64.92 20.15c 69.14
S.S + S.m.c +
T4 7.50 c 68.35 13.75c 62.01 16.2 c 67.53 19.05c 70.82
T. h +F. o
T5 C +F. o 6.2 c 73.6 12.4c 65.5 16.2 c 67.4 17.2c 73.6
Tomato plant
T6 0.0 c 100 0.0 d 100 0.0 d 100 0.0 d 100
alone
Overal Mean 10.71 19.11 24.27 29.41

F- test S S S S
S. Ed. (±) 5.709 5.240 4.808 4.659
C. D.(P =0.05) 12.16 11.16 10.24 9.92

Carbendazim = C

70
Disease intensity (%) 60
50
40
30
20
10
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Trichoderma harzianum on the disease intensity (%) of Fusarium oxysporum f.sp.

oxysporum

120
Reduction (%) 100
80
60
40
20
0
T0 T1 T2 T3 T4 T5 T6
Treatments
Trichoderma harzianum on the Reduction (%) of Fusarium oxysporum f.sp.

oxysporum

Similar findings have been reported by Hend et al. (2011) reported that
treatments of Trichoderma harzianum gave maximum control of wilt disease as
compared to other treatments. John et al. (2010) revealed that seed treatment with soil
application of Trichoderma spp were Foundmore effective in enhancing the growth and
suppress the wilt disease severity and also seed treatment plus soil application of
Trichoderma virens was most effective and significantly improved the plant growth.
Ramezani (2010) reported the mycoparasitism inhibitory effects of five Trichoderma
species (T. harzianum, T. koningi, T. longiconis, T. hamatum and T. viride) on the
growth of Fusarium oxysporum f.sp. lycopersici. In the greenhouse, seed treatment with
T. harzianum spores did not cause disease decrease but soil treatment with T. harzianum
spores caused disease decrease by 92%. Srivastava et al. (2010) reported that the
combination of fluorescent Pseudomonas, T.harzianum and arbuscular mycorrhizal
fungus (AMF) provided significantly better control than uninoculated treatment,
reducing disease incidence and severity by 74% and 67% in pots and field, respectively
. Bokhari and Kahkashan, (2012) reported that T. harzianum caused 55.5% reduction
in disease incidence while T. viride reduced root rot by 41.1%. The results obtained here
clearly indicate that T. harzianum is more effective antagonist against F. solani as
compared to T. viri. Hibar et al. (2007) reported that Trichoderma harzianum have
significantly reduced disease incidence especially when they were applied one week
before inoculation with the pathogen.
Kumar et al. (2007) reported that all critical appraisal of treatments revealed that
there is an effective control of chili with seedling dip in carbendazim (0.1%) and
drenching of plants at flowering stage either with carbendazim (0.1%) or mancozeb
(0.25%)
The probable reasons for such findings may be that Suwan et al., (2000) stated
that T. harzianum can produce peptibols namely Trichotoxins that could inhibit plant
pathogen and also promote plant growth. Kucuk and Kivanc (2002) stated T.
harzianum isolated from soil could inhibit F.oxysporum and produced chitinase to
degrade cell wall of tested plant pathogen. The soil solarization can be an effective tool
for management of Fusarium wilt on lettuce, especially when used within an integrated
program in conjunction with existing disease management tactics (Matheron and
Martin, 2010).

60 day
T1 T3 T4
150 days
T1 T3 T4
Plate 4.9: (T1) solarized soil + F.o, (T3) Trichoderma harzianum+ F.o and (T4)
Spent mushroom compost + Trichoderma harzianum+ F.o

60 day
T1 T3 T5
150 days
T1 T3 T5
Plate 4.10: (T1) solarized soil + F.o, (T3) Trichoderma harzianum + F.o and (T5)
Carbendazim + F.o

60 day
T1 T2 T4 T5
150 days
T1 T2 T4 T5
Plate 4.11: (T1) solarized soil + F.o, (T2) spent mushroom compost + F.o, (T4)
spent mushroom compost + Trichoderma harzianum + F.o and (T5)
Carbendazim + F.o

60 day
T0 T1 T3 T3 T4 T5 T6
150 days
T0 T1 T2 T3 T4 T5 T6
Plate 4.12: (T0) F.o alone, (T1) solarized soil + F.o (T2) spent mushroom
compost + F.o, (T3) Trichoderma harzianum+ F.o, (T4) spent mushroom
compost + Trichoderma harzianum+ F.o, (T5) Carbendazim + F.o and (T6)
Tomato plant alone

Chapter - V
SUMMARY AND CONCLUSION
Fusarial wilt caused by Fusarium oxysporum f.sp. lycopersici is a destructive

disease of tomato crop worldwide . This disease is caused by a soil borne fungal
pathogen that infects plants through roots at all stages of plant growth and is a major
problem in tomato crop in the warm, moist tropical regions of the world, causing
damping-off in nursery seedlings as well as stem rot, wilting and blight in adult plants
with consistent loss of production. The pathogen enters through the roots of the plant
and proliferates in the vascular tissues leading to breakdown of the water economy of
the infected plants. The disease causes great losses, on the susceptible varieties of
tomato especially when soil and air temperature are rather high during the warm season.
Typical symptoms of the disease are yellowing and wilting of leaves progressing
upward from the base of the stem. Initially, only one side of a leaf mid rib, one branch,
or one side of a plant is affected. The symptoms soon spread to the rest of the plant and
finally kill it.
Use of fungicides is quite uneconomical and it’s often leads to atmospheric
pollution, development of resistance in the pathogen also the risk of health hazards to
human being. Use of alternative methods like IDM against wilt disease is cheap and,
ecofriendly.
Integrated disease management (IDM), which combines biological, cultural,
physical and chemical control strategies is a holistic way rather than using a single
component strategy proved to be more effective and sustainable.
Neem pesticides play a vital role in pest management and hence have been widely
used in agriculture. Azadirachtin is the main ingredient used to manufacture bio
pesticides. Neem oil and seed extracts are known to possess germicidal and anti-
bacterial properties which are useful to protect the plants from different kinds of pests.
One of the most important advantages of neem-based pesticides and neem insecticides
is that they do not leave any residue on the plant. Moreover, in ayurveda, unani and
homeopathic medicine almost every part of the tree including seeds, leaves, roots, bark,
trunk and branches has multiple uses.
MASSOLC OS YUSYRAMMUS 120

The use of compost extracts to control diseases in plants is an effective alternative
to the use of pesticides .The abiotic component of compost is an important factor for its
suppressive effect in soils and considered a source of biocontrol agents against several
serious plant pathogens. The production of fungitoxic compounds such as organic acids
or ammonia from some organic amendments could enhance the antagonistic mechanism
involved in suppressing soil-borne fungi. Spent mushroom compost has physical and
chemical characteristics that make it ideal for blending with landscape mulch to
enhance growth of horticultural plants. Incorporation of composted Spent Mushroom
Substrate (SMS) not only improves the nutrient status but also neutralizes the acidity of
soils and facilitates cultivation even in problem soils. In addition, SMS also possesses
good bio-control activity against certain and soil borne diseases.
Several biocontrol agents including Pseudomonas fluorescens and Trichoderma
harzianum have been exploited in the management of soil-borne diseases. Several
bacterial and fungal antagonistic strains have been shown to reduce the damage caused
by different pathogens, including F. oxysporum f. sp. lycopersici .
The genus, Trichoderma is the most common saprophytic fungi and found in
almost any soil. The mycoparasite ability of Trichoderma species against some
economically soil borne plant pathogens. Trichoderma spp. commercial biological
control products are used in field crop and greenhouse system. These products are
known to control numerous soil-borne diseases such as wilt.
Pseudomonas spp. are common soil bacteria easily cultured from most
agricultural soils and plant rhizospheres. They have been studied in considerable detail
because of their ability to promote plant growth, either by directly stimulating the plant
or by suppressing pathogens.
Soil solarization is a natural hydrothermal process of disinfesting soil of plant pests and
pathogens that is accomplished through passive solar heating. The most well-known function of
solarization is reduction of soilborne inoculum of plant pathogens including fungi, bacteria, and
nematodes by direct thermal inactivation which is achieved at soil temperatures ranging from
40°C to more than 60°C. Many efforts are being made by several workers to improve
solarization efficiency in controlling soilborne pathogens including integration with a biological
control agent, lower dosages of chemical fungicides, organic amendments and physical
methods.

Hence, an experiment entitled, “Integrated Disease Management against
Fusarium oxysporum f.sp. lycopersici on Tomato (Lycopersicon esculentum L.)’’
was undertaken with the following objectives-
 To carry out pathogenicity test of Fusarium oxysporum f.sp. lycopersici by

following Koch’s postulate.
 To evaluate the effect of solarized soil with Neem cake and Carbendazim on
Fusarium oxysporum f.sp. lycopersici and plant growth parameters of
tomato.
 To evaluate the effect of carbendazim and solarized soil with Pseudomonas
fluorescens, Trichoderma harzianum, spent mushroom compost on
Fusarium oxysporum f.sp. lycopersici and plant growth parameters of
tomato.
The soil infested with culture of Fusarium oxysporum f.sp. lycopersici was
uniformly incorporated into the top 45 cm of 6 microplots. To verify that Fusarium
oxysporum f.sp. lycopersici had been established successfully, microplots were planted
with seeds of the wilt-susceptible tomato cultivar CO - 3. After, maximum percentage
of wilt occurred in all infested plots, while no wilt occurred in uninfested two
microplots. The aboveground plant material was cut off at the soil line and removed. On
2nd May 2013, each microplot was light irrigated and covered with an ultraviolet
stabilized, 30 µm clear polyethylene film in 4 microplots. The plastic was removed
thrice from four microplots at 15 days of interval that is up to June 2013, two microplots
was kept as nonsolarized soil.
The solarized and unsolarized soil was mixed with FYM @ 100 g / pot, the
bioagents Trichoderma harzianum and Pseudomonas fluorescens were applied @ 2 g /
pot, carbendazim 50 % W.P was applied @ 2 kg a.i / ha, whereas Neem cake powder was
amended in 10 pots with @ 10 g/ pot and spent mushroom compost soil in the pots @
20 g / kg of soil, four days before sowing the seeds of tomato variety (CO-3) . Seven
days after germination, four seedlings per pot were maintained in each treatment.
The treatments of solarized soil with Neem cake and Carbendazim revealed that
Neem cake with Carbendazim significantly increased the shoot length as compared with
other treatments at 30, 60 ,90,120 and 150 days after germination. At 150 days after
germination the fresh shoot weight of the treatments T2 (solarized soil + neem cake+ F.o

) and T3 (solarized soil + carbendazim+ F.o) significantly increased from T4 (solarized
soil + neem cake + carbendazim+ F.o) , T5 (carbendazim + F.o) , T0 ( F.o alone) and T1
(Solarized soil + F.o). At 161 days after germination the dry shoot weight increased in
T2, T3 , T4, T5 as compared with T0 and T1. At 150 days after germination the root length
in T2 and T3 significantly increased from T4, T5, T1 and T0. At 60, 90,120 and 150 days
after germination the disease intensity (%) was significantly reduced in T3 , T4 , T5 and
T2 as compared with T1 and T0.
The treatments of solarized soil with Pseudomonas fluorescens and spent

mushroom compost revealed that at 30 days after germination the shoot length was
significantly increased in T5 (carbendazim + F.o, 20.97 cm) as compared with other
treatments. At 60 days after germination the shoot length was significantly increased in
the treatments of T5 (carbendazim + F.o, 65.75 cm) and T4 (solarized soil +
Pseudomonas fluorescens + spent mushroom compost, 58.05 cm) as compared with
other treatments. At 90, 120, and150 days after germination the shoot length was
significantly increased in T4 as compared with T1 and T0. Fresh and dry shoot weight (g)
was found significantly increased in T4 (solarized soil + spent mushroom compost +
Pseudomonas fluorescens), T5 (carbendazim + F.o), T3 (solarized soil + Pseudomonas
fluorescens) and T2 (solarized soil + spent mushroom compost) as compared with T1,
T0. At 150 days after germination the root length was significantly increased in the
treatments T4, T3 and T2 from T5, T1 and T0. Fresh root weight was significantly
increased in T5, T2, T4 and T3 as compared with T1 and T0. At 60 and 90 days, the
disease intensity (%) of Fusarium oxysporum in the treatments T5, T4, T3 and T2 are
significantly reduced as compared with T1, T0. At 120 and 150 days significantly
reduced the disease intensity was found in T5 (16.2, 17.2%) as compared with T0 (49.9,
65.3%) respectively.
The treatments of solarized soil with Trichoderma harzianum and spent

mushroom compost revealed that at 30 and 60 days after germination the shoot length of
tomato in T5 (carbendazim+ F.o) significantly increased from T3 (solarized soil +
Trichoderma harzianum+ F.o) , T2 (solarized soil + spent mushroom compost+ F.o) ,
T1 ( solarized soil + F.o) and T0 (F.o alone). At 90 days there was significant increase
in the shoot length (cm) in the treatments T6 (Tomato plant alone, 108.00 cm), as
compared with T3, T5, T2, T1 and T0. At 120 and 150 days, shoot length was
significantly increased in T6 (Tomato plant alone) as compared with T5, T3, T2, T1 and

T0. The fresh shoot weight was significantly increased in the treatments T4, T3 and T5
from T1 and T0. Root length of tomato was significantly increased in T4 and T3 from T5,
T1, and T0. The fresh and dry root weights were significantly increased in T4, T3, T5 and
T2 as compared with T1 and T0. At 60, 90,120,150 days, the disease intensity (%) of
Fusarium oxysporum in the treatments T5, T4, T3 and T2 significantly reduced as
compared with T0.
The yield of tomato plants treated with Neem cake / Spent mushroom compost /
Trichoderma harzianum / Pseudomonas fluorescens with carbendazim was found non
significant from untreated plants without fusarium but significantly increased as
compared without carbendazim treated plants.
CONCLUSION
From the findings it’s concluded that IDM of Fusarium oxysporum f.sp.
lycopersici on tomato using Carbendazim ,biology method (Pseudomonas fluorescens
and Trichoderma harzianum ,Neem cake and spent mushroom compost ) and physical
method (Soil solarization) and chemical method shows significantly minimized the
incidence of Fusarium oxysporum f.sp. lycopersici and improved the growth and the
yield of tomato grown in infected plot .The treatment combination of Pseudomonas
fluorescens and spent mushroom compost with solarized soil showed most effective in
reduction the disease intensity (%) of Fusarium oxysporum f.sp. lycopersici and
increased the growth of tomato plants.

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Disease intensity after 60 days A-
ANOVA :
Source d. f. S.S. M.S.S. F. Cal. Result
F.Tab.5%.
Treatment 6 2292.411 382.0685 6.8466667 2.45 S
Error 28 1562.500 55.8036 - - -
TOTAL 34 - - - -
ANOVA:
Source d. f. S.S. M.S.S. F. Cal. F.Tab.5% Result
Treatment 6 5297.375 882.8959 17.58723 2.45 S
Error 28 1405.627 50.2010 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 10762.618 1793.7696 56.967674 2.45 S
Error 28 881.650 31.4875 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 17530.025 2921.6709 117.45495 2.45 S
Error 28 696.495 24.8748 - - -
TOTAL 34 - - - -
APPENDIX i
Disease intensity after 60 days B-
ANOVA :
Treatment 6 2026.786 337.7976 3.7366255 2.45 S
Error 28 2531.250 90.4018 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 4919.643 819.9405 11.849462 2.45 S
Error 28 1937.500 69.1964 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 9999.671 1666.6119 31.297351 2.45 S
Error 28 1491.025 53.2509 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 16206.395 2701.0659 42.112035 2.45 S
Error 28 1795.920 64.1400 - - -
TOTAL 34 - - - -
APPENDIX ii
Disease intensity after 60 days C-
ANOVA :
Treatment 6 2060.268 343.3780 4.2146119 2.45 S
Error 28 2281.250 81.4732 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 5026.786 837.7976 12.205962 2.45 S
Error 28 1921.875 68.6384 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 10363.861 1727.3101 29.89257 2.45 S
Error 28 1617.950 57.7839 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 16655.184 2775.8640 51.147124 2.45 S
Error 28 1519.620 54.2721 - - -
TOTAL 34 - - - -
APPENDIX iii
Shoot length after 30 days A
ANOVA :
Treatment 6 1227.001 204.5001 27.147907 2.45 S
Error 28 210.919 7.5328 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 12448.021 2074.6702 54.960752 2.45 S
Error 28 1056.950 37.7482 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 32514.668 5419.1113 74.157305 2.45 S
Error 28 2046.125 73.0759 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 25847.175 4307.8626 36.547103 2.45 S
Error 28 3300.403 117.8715 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 25661.095 4276.8492 21.572633 2.45 S
Error 28 5551.097 198.2535 - - -
TOTAL 34 - - - -
APPENDIX iv
Shoot length after 30 days B
ANOVA :
Treatment 6 463.399 77.2331 10.846293 2.45 S
Error 28 199.379 7.1207 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 7051.843 1175.3071 23.239306 2.45 S
Error 28 1416.075 50.5741 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 28603.311 4767.2185 71.938624 2.45 S
Error 28 1855.500 66.2679 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 22372.297 3728.7162 44.208519 2.45 S
Error 28 2361.628 84.3438 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 23327.975 3887.9959 30.455296 2.45 S
Error 28 3574.547 127.6624 - - -
TOTAL 34 - - - -
APPENDIX v
Shoot length after 30 days C

Treatment 6 477.120 79.5200 9.1404677 2.45 S
Error 28 243.594 8.6998 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 6263.986 1043.9976 20.610906 2.45 S
Error 28 1418.275 50.6527 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 30920.468 5153.4113 61.108507 2.45 S
Error 28 2361.300 84.3321 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 24997.031 4166.1719 39.084874 2.45 S
Error 28 2984.603 106.5929 - - -
TOTAL 34 - - - -
ANOVA :
Treatment 6 25671.810 4278.6351 26.858845 2.45 S
Error 28 4460.422 159.3008 - - -
TOTAL 34 - - - -
APPENDIX vi
Fresh shoot weight after 150 days A-
ANOVA :
Treatment 6 29099.486 4849.9143 7.6749125 2.45 S
Error 28 17693.700 631.9179 - - -
TOTAL 34 - - - -
Fresh shoot weight after 150 days B-
ANOVA :
Treatment 6 26194.696 4365.7827 11.45244 2.45 S
Error 28 10673.875 381.2098 - - -
TOTAL 34 - - - -
Fresh shoot weight after 150 days C-
ANOVA :
Treatment 6 26462.200 4410.3667 13.635051 2.45 S
Error 28 9056.825 323.4580 - - -
TOTAL 34 - - - -
Dry shoot weight after 161 days A-
ANOVA :
Treatment 6 11653.571 1942.2619 9.3913739 2.45 S
Error 28 5790.775 206.8134 - - -
TOTAL 34 - - - -
Dry shoot weight after 161 days B-
ANOVA :
Treatment 6 11382.693 1897.1155 11.568596 2.45 S
Error 28 4591.675 163.9884 - - -
TOTAL 34 - - - -
APPENDIX vii
Dry shoot weight after 161 days C-
ANOVA :
Treatment 6 11168.300 1861.3833 #VALUE! 2.45 #VALUE!
Error 28 #VALUE! #VALUE! - - -
TOTAL 34 - - - -
Root length after 150 days A-
ANOVA :
Treatment 6 1814.993 302.4988 17.261867 2.45 S
Error 28 490.675 17.5241 - - -
TOTAL 34 - - - -
Root length after 150 days B-
ANOVA :
Treatment 6 1639.311 273.2185 25.596375 2.45 S
Error 28 298.875 10.6741 - - -
TOTAL 34 - - - -
Root length after 150 C-
ANOVA :
Treatment 6 1873.346 312.2244 15.946524 2.45 S
Error 28 548.225 19.5795 - - -
TOTAL 34 - - - -
Fresh root weight after 150 days A-
ANOVA :
Treatment 6 131.505 21.9174 8.3508538 2.45 S
Error 28 73.488 2.6246 - - -
TOTAL 34 - - - -
APPENDIX viii
Fresh root weight after 150 days B-
ANOVA :
Treatment 6 126.460 21.0767 31.452666 2.45 S
Error 28 18.763 0.6701 - - -
TOTAL 34 - - - -
Fresh root weight after 150 days C-
ANOVA :
Treatment 6 135.290 22.5483 16.4145 2.45 S
Error 28 38.463 1.3737 - - -
TOTAL 34 - - - -
Dry root weight after 161 days A-
ANOVA :
TOTAL 34 - - - -
Dry root weight after 161 days B-
ANOVA :
TOTAL 34 - - - -
Dry root weight after 161 days C-
ANOVA :
Treatment 6 32.211 5.3685 10.203878 2.45 S
Error 28 14.731 0.5261 - - -
TOTAL 34 - - - -
APPENDIX ix
Yield / plant (g) after 150 days A-
ANOVA:
F.
Source d. f. S.S. M.S.S. F. Cal. Tab.
Result
5%
Treatment 6 S
159417.489 26569.5815 61.491653 2.45
Error 28 12098.362 432.0844 - - -
TOTAL 34 - - - -
Yield / plant (g) after 150 days B-
ANOVA :
F.
Source d. f. S.S. M.S.S. F. Cal. Tab. Result
5%
Treatment 6 S
130097.645 21682.9408 93.303346 2.45
Error 28 6506.973 232.3919 - - -
TOTAL 34 - - - -
Yield / plant (g) after 150 days C-
ANOVA :
F.
Source d. f. S.S. M.S.S. F. Cal. Tab. Result
5%
Treatment 6 S
137641.124 22940.1873 135.2239 2.45
Error 28 4750.087 169.6460 - - -
TOTAL 34 - - - -
APPENDIX x
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Integrated Disease Management against Fusarium oxysporum f.sp. lycopersici

Thesis · September 2015

IDM against fusarium wilt View project

Comparison of Tomato varieties View project

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HUSSEIN ALI SALIM

ID. No. 12PHCPP103

FACULTY OF AGRICULTURE, SCHOOL OF AGRICULTURE

It is certified that the thesis entitled ‘‘Integrated Disease Management against

Place: Allahabad Prof. (Dr.) Sobita Simon

This thesis entitled ‘‘Integrated Disease Management against Fusarium oxysporum

Name Evaluation Signature

2. Dr. Abhilasha A. Lal

3. Dr. Shafaat Ahmad

4. Dr. Sunil Zacharia

This is to certify that I have personally worked on the research entitled

Date: HUSSEIN ALI SALIM

Place: ALLAHABAD ID. No. 12PHCPP103

Tomato (Lycopersicon esculentum Mill.) is an important vegetable crop belongs

Table. 1.1: Major Tomato producing Countries in the world

Source: Indian Horticulture Database, 2011

Table.1.2: The Area, Production and Productivity of Tomato in India

Year Area (000’ha) Production(000’tons) Productivity(tons/ha)

2006-2007 596.0 10055.0 16.9

2007-2008 566.0 10303.0 18.2

2008-2009 599.0 11149.0 18.6

2009-2010 634.4 12433.2 19.6

2010-2011 865.0 16826.0 19.5

Source: Indian Horticulture Database, 2011

Integrated disease management (IDM)

Soil solarization is a natural hydrothermal process of disinfesting soil of plant

Carbendazim, one of the fungicide was approved by International Standardization

In view of the above the present investigation “ Integrated Disease Management

1- To carry out pathogenicity test of Fusarium oxysporum f.sp. lycopersici by

Fusarium oxysporum f. sp. lycopersici

Shishido et al. (2005) examined the efficiency of nonpathogenic Fusarium oxysporum

Nirmaladevi1 and Srinivas (2012) isolated Fusarium oxysporum f. sp. lycopersici

Gnanamanickam et al. (2002) reported that successful biological control systems

Montealegre et al. (2005) evaluated Trichoderma harzianum 650 (Th650) and

Ramezani (2010) observed the mycoparasitism inhibitory effects of five Trichoderma

Mwangi et al. (2011) investigated the ability of an isolate of Trichoderma harzianum

Alwathnani and Kahkashan (2012) observed that in dual culture technique

Srinivasan et al. (2009) evaluated bacteria strains (Pseudomonas putida FC-6B,

Ardebili et al. (2011) evaluated Pseudomonas fluorescens CHA0 against Fusarium

Meenakshisundaram and Santhaguru (2012) screened fluorescent Pseudomonas spp.

Saravanan et al. (2013) focused on the antagonistic potential of fluorescent

Tayal et al. (2011) investigated the effectiveness of Glomus fasciculatum, Trichoderma

Stapleton (2000) reported that Solarization occurs through a combination of physical,

Rajput et al. (2006) conducted pathogenicity test of Fusarium oxysporum f. sp.

Amini and Dzhalilov (2010) evaluated six fungicides; benomyl, carbendazim,

The present study entitled “Integrated Disease Management against Fusarium

3.2. Topographical conditions:

3.3. Cleaning and sterilization of glass wares:

3.4. Preparation of media (PDA):

AARITEA MLA ALAIRETAM 39

3.5. Preparation of Czapek's media (CDA):

For re-isolation of Fusarium oxysporum f.sp. lycopersici from mother culture in

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Plate: 3.1 Isolation and purification of Fusarium oxysporum f.sp. lycopersici

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Plate: 3.3 Pure culture of Fusarium oxysporum f.sp. lycopersici on Czapek's

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