Vegetables Root Rot Disease Management by an Integrated ... 3/Issue 5/IJEIT1412201311_07.pdf · vegetables under protected cultivation system in ARE were subjected to survey of either

ISSN: 2277-3754

ISO 9001:2008 Certified International Journal of Engineering and Innovative Technology (IJEIT)

Volume 3, Issue 5, November 2013

40

Abstract — The most reported areas for high production of

vegetables under protected cultivation system in ARE were

subjected to survey of either root or shoot systems fungal diseases

at two growth stages of plant growth. Surveyed vegetable crops,

i.e. Cucumber, Pepper, Tomato showing damping-off, root rot

and wilt symptoms were subjected to isolation trails for the

purpose of isolation the causal organisms. Rhizospheric samples

of different healthy and diseased plants were collected at

flowering growth stage from the same surveyed plastic houses

distributed for determining the frequency occurrence of different

fungi and bacteria associated with the root region of healthy and

infected roots of cucumber, pepper, tomato and cantaloupe

plants. The root fungal pathogens isolated from rhizospheric soil

were Pythium spp., Fusarium spp. Rhizoctonia solani,

Macrophomina sp. Sclerotinia spp., Sclerotium rolfsii which

recorded in high frequency. Different approaches of some

antagonistic fungal, bacterial and yeast agents applied as seed

treatment or soil drench was evaluated against various soil-borne

pathogens causing vegetables root rot disease under greenhouse

conditions. The tested pathogenic fungi were Alternaria solani

Fusarium solani, F. oxysporum, Rhizoctonia solani, Sclerotium

rolfsii, Macrophomina phaseolina and Pythium sp., meanwhile

the tested bio-agents were Trichoderma harzianum, T. Viride and

Bacillus subtilis, Pseudomonas fluorescens and Sacchromyces

serevisiae. Significant reduction in the disease incidence was

observed in bio-agent treatments in comparison with untreated

control. Root rot incidence, at pre-emergence stage, significant

effect was observed in bio-agent treatments as seed soaking

comparing with soil drench treatment. The treated seeds showed

a protective effect for seeds germination against the invasion by

soil-borne pathogenic fungi. Meanwhile, soil drenched with

different bio-agents showed more efficacy for reducing root rot

incidence at post-emergence growth stage of tested vegetables,

Cucumber, Cantaloupe, Tomato and Pepper. Under greenhouse

conditions soil drench with furfural, Humic & Folic acid and/or

bio-agents treatments were evaluated against root rot incidence

of Cucumber, Cantaloupe, Tomato and Pepper in pot

experiments. Applied treatments of the bio-agents in combination

with the tested chemicals resulted in higher significant reduction

in root rot incidence than each of them alone. Treatments of T.

harzianum either alone or combined with chemicals were

superior for reducing root rot disease for all tested vegetable

plants followed by B. subtilis treatments comparing with the other

tested ones. Moreover, the efficacy of Calcium chloride, Thyme

oil and /or bio-agents as seed dressing against disease incidence

was evaluated in pot experiments under artificially infested with

vegetables root rot causal organisms under greenhouse

conditions. All applied treatments reduced significantly root rot

incidence at both pre-, and post-emergence growth stages of

Cucumber, Cantaloupe, Tomato and Pepper plants comparing

with untreated check control. The obtained results showed that

combination treatments of Calcium chloride, Thyme oil with bio-

agents reduced significantly root rot incidence of all grown

vegetables comparing with the application of each of them alone.

Also, in pot experiment the introduced bio-agents proved to

continue keeping their antagonistic effect against pathogenic

fungi for over one cultivation season resulted in minimize the

incidence of root rot disease at both pre-, and post-emergence

plant growth stages. Meanwhile, under plastic houses conditions,

the efficacy of different plant resistance inducers and/or bio-

agents treatments against root diseases incidence of some

vegetables were evaluated under plastic houses conditions. The

evaluated treatments were applied as soil drench before

transplanting at commercial plastic houses. The addition of a

biological control agent in combination with plant resistance

inducers resulted in increased symptom less plant stand over the

biological agent. These methods characterized as

environmentally safe, bioactive natural products which able

successfully to control phytopathogenic fungi in crop production

systems. The present review summarizes studies starting from

survey of detected infected vegetable crops grown under protected

cultivation system up to evaluate some control measures of

fungicides alternatives approaches, e.g. some plant resistance

inducers, essential oils and bio-control agents on the root rot

incidence of some vegetables under greenhouse and plastic house

conditions. This work was carried out during a project supported

by the Science and Technology Development Fund (STDF),

Egypt.

Index Terms—Bio-agents survival, biological control,

cantaloupe, cucumber, essential oils, greenhouse, pepper, plant

resistance inducers, plastic houses, root diseases, seed

treatment, soil drench, tomato.

I. INTRODUCTION

Protected agriculture is considered an important means of

increasing the productivity and quality of most vegetable

crops. Recently, there has been an increase in interest in

protected agriculture (PA) in Egypt. The demand for plastic

houses has increased and their use has spread in throughout

different regions, where there were protected houses

belonging to Governmental agro-research stations and

others belonging to public-sector cooperatives. Vegetable

crops are grown worldwide as a source of nutrients and fiber

Vegetables Root Rot Disease Management by

an Integrated Control Measures under

Greenhouse and Plastic Houses Conditions in

Egypt – A Review Nehal S. El-Mougy, M. M. Abdel-Kader, S. M. Lashin

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in the human diet. In Egypt, the most common cultivated

vegetables under PA system are Cucumber, sweet and hot

Pepper, Tomato, Cantaloupe and Eggplant [1]. Under plastic

houses conditions such vegetables are reported to be

infected with different root and foliar diseases, i.e. damping-

off, root rot, white rot, wilt, downy and powdery mildew

[2,3,4]. The impact of plant pests on the aspiring producer of

greenhouse vegetables is direct and significant. The

prospective producer must understand that the greenhouse

condition is a paradise for both the crop and the

accompanying pests that afflict it. Plastic houses have led to

changes in the microclimate of protected crops. Restricted

air exchange results in the atmospheric humidity being

much higher inside insulated greenhouses than conventional

ones which encourage several plant diseases and cause

physiological disorders. Climate change could alter stages

and rates of development of the pathogen, modify host

resistance, and result in changes in the physiology of host-

pathogen interactions [5]. Fungal plant pathogens can cause

devastation in these crops under appropriate environmental

conditions. The challenges for producers in managing these

diseases are ever-increasing, as consumer demand for year-

round production of fresh vegetables with reduced or no

pesticide residues continues to grow. Concerns over the

potential impact of disease management practices including

the use of fungicides on the environment or on consumer

health have prompted producers to examine alternative

methods to combat fungal diseases. There is a growing need

to develop alternative approaches for controlling plant

diseases. Root and stem rot diseases caused by pathogens

which survive in soil are responsible for serious losses in

vegetables crop yield, e.g. Tomato [6], Cantaloupe [7] and

Pepper [8]. It was also, recorded that [9] Fusarium stem and

root rot of cucumber was observed at four commercial

greenhouses in Leamington, Ontario, Canada causing losses

of 25-35%. Vegetable producers confronted with the

challenges of managing fungal pathogens have the

opportunity to use fungi, bacteria and yeasts as biological

control agents. Several commercially available products

have shown significant disease reduction through various

mechanisms to reduce pathogen development and disease.

Plant diseases need to be controlled to maintain the quality

and abundance of food, feed, and fiber produced by growers

around the world. Different approaches may be used to

prevent, mitigate or control plant diseases. Beyond good

agronomic and horticultural practices, growers often rely

heavily on chemical fertilizers and pesticides. Such inputs to

agriculture have contributed significantly to the spectacular

improvements in crop productivity and quality over the past

100 years. However, the environmental pollution caused by

excessive use and misuse of agrochemicals, as well as fear

mongering by some opponents of pesticides, has led to

considerable changes in people’s attitudes towards the use

of pesticides in agriculture. Today, there are strict

regulations on chemical pesticide use, and there is political

pressure to remove the most hazardous chemicals from the

market. Additionally, the spread of plant diseases in natural

ecosystems may preclude successful application of

chemicals, because of the scale to which such applications

might have to be applied. Consequently, some pest

management researchers have focused their efforts on

developing alternative inputs to synthetic chemicals for

controlling pests and diseases. Among these alternatives are

those referred to as biological control. The application of

biological controls using antagonistic microorganisms has

proved to be successful for controlling various plant

diseases in many countries [10]. However, this is not an

easy method, and it is costly to apply; however it can serve

as the best control measure under greenhouse conditions.

Trichoderma harzianum introduced to the soil, was able to

reduce root rot incidence of faba bean plants significantly

more than the fungicide Rizolex-T [11]. In recent years,

several attempts have been made to overcome this obstacle

by applying antagonistic microorganisms. Trichoderma spp.

are well documented as effective biological control agents of

plant diseases caused by soil-borne fungi [12, 13, 14]. Many

investigators [15,16] observed that the application of wheat

bran colonized by T. harzianum to soil infested with R.

solani and S. rolfsii, reduced the incidence of root diseases

caused by these pathogens. As for antagonistic bacteria [17]

found that seed treatment with Bacillus spp. was actively

controlled three fungal root diseases of wheat. Also,

Pseudomonas cepacia or Pseudomonas fluorescens applied

to pea seeds act as biological control agent against Pythium

damping-off and Aphanomyces root rot and was able to

reduce diseases incidence [18, 19]. Considerable researches

has been done to investigate antagonistic microbes for use in

seed treatments as reported by several workers

[20,21,22,23]. In this regard, many crops are susceptible to

seed and seedling root rot caused by soil-borne fungi or by

pathogens carried on the seed. Biological seed treatments

may provide an alternative to chemical control of many soil

and seed-borne pathogens. Bio-priming, a seed treatment

system that integrates the biological and physiological

aspects of disease control, involves coating the seed with

fungal or bacterial bio-control agents. Furthermore, soil

drench with T. harzianum was significantly able to reduce

the incidence of bean root rot of bean and pepper wilt

diseases [11,24]. Furthermore, with the knowledge of the

adverse effects of synthetic fungicides worldwide, attention

is rapidly, being shifted to non-synthetic, safer alternatives.

The present review focuses on finding compounds that are

safe to humans and the environment, e.g. chemical

resistance inducers and/ or bio-agents. In this regards, plant

products are characterized as having a wide range of volatile

compounds could be used as alternative antibacterial and

antifungal treatments [25]. It is evident from reviews by

several investigators that Humic and Fulvic acids have been

early recorded to have appositive effect against plant

pathogens and their cells biological activities [26,27,28]. On

the other hand, furfural is a naturally occurring compound,

and recently used as a new pesticide active ingredient

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intended for the use as a fumigant to control root infesting

plant parasitic nematodes and fungal plant diseases.

Moreover, [29] reported that most of drip irrigation

treatments reduced populations of Pythium ultimum and F.

oxysporum and increased stem height compared with the

nontreated controls. Metham sodium, furfural + metham

sodium, sodium azide, and chloropicrin significantly

reduced the incidence of Liatris stem rot caused by

Sclerotinia sclerotiorum. Moreover, the concern of

pesticides use with respect to human health and environment

has brought increasing interest in alternatives use by

avoiding negative effect on the environment. Essential oils

are known for their natural components, such as mono

terpenes, diterpenes, and hydrocarbons with various

functional groups. Many other researchers have reported

antifungal activities [30,31,32] of essential oils in food

applications, pharmaceutical research and other areas as

plant disease control. The present article reviewed surveying

the root diseases infection and associated rhizospheric

microorganisms of some vegetables grown under protected

cultivation system at different Governmental agro-research

stations plastic houses located at different locations

throughout Egypt. Moreover, the review focuses on

recorded compounds that are safe to humans and the

environment, e.g. some plant resistance inducers, essential

oils as well as bio-control agents which may provide an

alternative control of many soil and seed-borne pathogens.

The objective of the present work was to evaluate fungicide

alternatives and /or bio-agents against root rot incidence

when used as seed and soil treatment under greenhouse and

plastic house conditions. Survival and activity of introduced

bio-agents to the soil was also considered.

II. SURVEYING OF VEGETABLE FUNGAL

DISEASES THROUGHOUT DIFFERENT

PROTECTED CULTIVATION LOCATIONS

The most reported areas for high production of vegetables

under protected cultivation system in ARE were subjected to

survey of either root or shoot systems diseases at two

growth stages of plant growth, i.e seedling (30-60 days after

transplanting) and maturity (80-120 days after

transplanting). The percentage of different diseases

incidence was recorded at different commercial greenhouses

distributed in five governorates, i.e. Giza, Cairo, Kalubia,

Ismaelia and Behiera. The average percentages of root and

foliar diseases infections were calculated as the number of

infected plants in relative to the total number of examined

plants. Crop monitoring is the continually on-going

surveillance to detect the presence of a pest or disease at the

very early stages of development of the disease or pest

population, before economic damage has occurred.

Therefore, the research team involved in working the crop

monitoring are enough qualified of the common disease

problems and what to look for to detect the presence of

disease symptoms in certain crop.

The recorded diseases of surveyed different vegetable

crops, i.e. Cucumber, Pepper, Tomato and Cantaloupe

grown under PA in different governorates in Egypt [33]

were damping-off, root-rot, white rot and wilt. The obtained

results revealed that the surveyed plants at early stages (30-

60 days after transplanting) showed root infections

expressed at highest records with wilt infection followed by

root-rot and damping-off. Cantaloupe infection with

damping-off, root rot and wilt diseases was only recorded at

plastic houses located at Ismaelia governorate. It is observed

that the recorded data indicate that all cultivars of various

surveyed vegetable crops grown under protected cultivation

system are susceptible to disease infection with both soil-

borne and airborne plant pathogens at all surveyed locations.

In this regard, it was recorded that the most important root

diseases in greenhouses systems are caused by fungi of

various species of Pythium and Phytophthora. These fungi

are known collectively as water moulds and are important

pathogens in soils in field [3]. Moreover, disease organisms,

insects, and nematodes can cause serious problems in plastic

houses. Without a real winter period, populations of pests

continue to build, and many are sustained throughout the

year. With this mild climate comes the adaptability of both

temperate and tropical pests, thus presenting a large number

of potential problems for greenhouse crops [2]. These

reports are in a harmony with the present results of our

study. Surveyed production areas were examined for the

initial symptoms of disease during routine crop maintenance

activities, whereas, the earlier a disease is found and

identified. The most recorded common diseases affecting

greenhouse vegetables were also previously reported in

Florida [3,4,34,35,36].

III. ISOLATION DIFFERENT SOILBORNE

MICROORGANISMS ASSOCIATED WITH

HEALTHY AND INFECTED VEGETABLES

Soil microorganisms, fungi and bacteria, associated with

the root region of healthy and infected roots of Cucumber,

Pepper and Tomato plants grown in plastic houses were

isolated [33]. They added that the obtained results showed

the frequency occurrence of different fungi in assayed

rhizospheric soil samples. The root fungal pathogens e.g.

Pythium spp., Fusarium spp. Rhizoctonia solani,

Macrophomina sp. Sclerotinia spp., Sclerothium rolfsii were

recorded in high frequency comparing with other fungal

genera Alternaria spp., Aspergillus spp., Penicillium spp.

and Trichoderma spp. The genus Fusarium represented in

highest records followed by the genus Rhizoctonia and

Sclerotium respectively at all assayed samples. Meanwhile,

the pathogens referred to genus Pythium, Macrophomina

and Sclerotinia were represented in a lesser frequency.

Vegetable crops, i.e. Cucumber, Pepper, Tomato showing

damping-off, root rot and wilt symptoms were subjected to

isolation trails for the purpose of isolation the causal

organisms. The isolated fungi were previously recorded as

the main causal of root diseases of surveyed vegetables

[37,38,39,40,41,42]. (Concerning the total bacterial counts,

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results [33] showed that the rhizosphere samples of

cucumber, pepper, tomato and cantaloupe were differed in

their total bacterial counts. Results also revealed that the

four main bacterial groups were common in most plants

rhizosphere. In rhizospheric samples collected from Giza

plastic houses showed a high records of bacterial count in

cucumber rhizosphere, compared to the bacterial count in

pepper and tomato samples, respectively. The isolated

bacteria are identified according to specific characteristics as

Bacillus sp., Agrobacerium sp. and non –fluorescent

Pseudomonas. In this regards, plant pathogenic bacteria are

dominant soil-borne microorganisms which cause many

serious diseases of plants throughout the world [43] causing

relatively damage and economic cost [44]. Survival of plant

pathogenic bacteria in nature occurs most commonly in

plant debris left on the soil surface, in and on seeds, in soil,

and in association with perennial hosts [45]. Knowledge of

survival is usually essential to intervene in dissemination

and for disease management. They added that Dissemination

commonly occurs by windblown soil and sand particles that

cause plant wounding, particularly during or after rains or

storms.

IV. SURVIVAL AND ANTAGONISTIC ACTIVITY OF

INTRODUCED BIO-AGENTS TO THE SOIL

Biological control of soil-borne plant diseases is growing

in importance as the demand for more environmental

friendly management strategies for plant pests increases.

The application of biological control using antagonistic

microorganisms proved to be successful for controlling

various plant diseases in many countries [10]. Fungi in the

genus Trichoderma are among the most widely

commercialized bio-control agents for soil-borne diseases of

crops [46,47,48]. Species of Trichoderma are present in

nearly all soils and other diverse habitats [49]. The ability of

several Trichoderma spp. isolates to control various plant

pathogens, such as Pythium spp., Fusarium spp.,

Rhizoctonia solani, Verticillium spp., Sclerotium rolfsii,

Botrytis cinerea, Armillaria spp., Botryoshaeria spp. and

others, has been demonstrated [12,13,14,50,51]. As for

antagonistic bacteria, [17] found that seed treatment with

Bacillus spp. was actively controlled three fungal root

diseases of wheat. Also, Pseudomonas cepacia or

Pseudomonas fluorescens applied to pea seeds act as

biological control agent against Pythium damping-off and

Aphanomyces root rot and was able to reduce diseases

incidence [18,19]. Also, Bacillus sp. gave a highly

antagonistic effect against some pathogenic fungi including

Fusarium solani [53]. It is critical that artificially introduced

inoculum is able to colonize, establish, compete and survive

in the complex soil environment. Satisfactory bio-control by

Trichoderma spp. is apparently dependent on the attainment

of some minimum threshold populations in the soil [54] and

the continued presence of living cells of the antagonist, as

well as a food base [16]. Artificial antagonist inoculum

tends to decline in natural soils, and could degenerate to

undetectable levels within a relatively short time [54].

Degeneration of active biomass may be accelerated by

edaphic factors, such as soil drying [55] or nutrient

depletion. Failure of antagonist inoculum to survive and

accord long term disease control is a major hindrance to the

adoption of biocontrol strategies in the management of soil-

borne plant diseases [56]. Conversely, indigenous

populations of phytopathogens are often well established

and adapted, and may persist in the soil for long periods,

from where they could multiply and cause disease. In

practice, booster applications of antagonists are often

necessary to prolong the biocontrol effect. Repeated

applications could significantly raise production costs. Pot

experiment was carried out in the open greenhouse of Plant

Pathology Dept., National Research Centre, Egypt for

evaluating the survival and activity of bio-control agents

which previously introduced to the soil, as soil treatments,

one season before comparing with fresh ones against root rot

disease incidence [57]. Cucumber, Cantaloupe, Tomato and

pepper plants were used in this experiment as a model of

widespread vegetable crops mainly grown under protected

cultivation system in Egypt and showed susceptibility to

attack with root rot pathogens [33]. They found that

incidence of pre-, and post-emergence root rot show that the

previously bio-agent applied treatments caused a significant

effect on root rot incidence at both plant growth stages of

grown vegetables comparing with control. At the first

cultivation season, pre-, and post-emergence stages

treatment with the bio-agent, T. harzianum caused the

highest protection for seeds germination against the invasion

by soil-borne pathogenic fungi followed by the others, T.

viride, B. subtilis and P. fluorescence, respectively in

descending order. The yeast, S. serevisiae showed the lowest

protection in this concern. Similar trend was observed at the

second cultivation growing season. Regarding the obtained

results in the second cultivation season and comparing them

with results obtained in the previous cultivation season, it

could be observed that survival of both pathogenic and

antagonistic microorganisms was proved. Although, the

percentage of root rot incidence in control check treatment

was lower in second cultivation season than the first one, the

bio-agent treatments also showed lesser disease incidence.

This phenomenon could be explained as the count of

introduced microorganisms into soil, either pathogenic or

antagonistic, facing the environmental conditions which

reflecting on their counts but not activity to reach their

establishment stage which called soil equilibrium

community. They stated that survival of both pathogenic and

antagonistic microorganisms was proved. Although, the

percentage of root rot incidence in control check treatment

was lower in second cultivation season than the first one, the

bio-agent treatments also showed lesser disease incidence.

This phenomenon could be explained as the count of

introduced microorganisms into soil, either pathogenic or

antagonistic, facing the environmental conditions which

reflecting on their counts but not activity to reach their

establishment stage which called soil equilibrium

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community. Therefore, the introduced bio-agents continue

keeping their antagonistic effect against pathogenic fungi for

over one cultivation season resulted in minimize the

incidence of root rot disease at both pre-, and post-

emergence plant growth stages. They conclude that the

introduced bio-agents continue keeping their antagonistic

effect against pathogenic fungi for over one cultivation

season resulted in minimize the incidence of root rot disease

at both pre-, and post-emergence plant growth stages.

Results reported by [57] are also confirmed by several

researchers [11,58,59]. Biological control of seedling

diseases using antagonistic fungi and bacteria has received

increasing attention. There are several methods for

introducing and delivering bio-agent to the cultivation soil.

In this regards, antagonists applied to seeds prior to planting

colonize the rhizosphere of seedlings and thus are present at

or near the pathogen’ infection court, where they act by

producing antifungal or antibiotic compounds, through

hyper parasitism, or by competitively colonizing sperm

sphere and rhizosphere substrates [60]. Seed treatment is an

attractive delivery system either fungal or bacterial bio-

protectants [61]. Bio-protectants applied to seeds may not

only protect seeds [12] but also may colonize and protect

roots [62]. On the other hand, [20] added a suspension of the

bacterium Pseudomonas fluorescens to 1.5% methyl

cellulose coated, surface sterilized sweet corn seed prior to

hydrating the seeds between moistened paper towels. The

two methods were used to protect tomato and sweet corn

against Pythium damping-off. In the study carried by [57]

the obtained results revealed long term antagonistic ability

against soil borne pathogens for over one cultivation season

through mixing bio-agents with cultivation soil. In this

concern it was reported that temporal population dynamics

and survival of antagonists are relevant for the management

of disease epidemic cycles arising from amplification of

pathogen inoculum. Facultative parasites are capable of

saprophytic multiplication, even in the absence of a crop,

hence increasing the amount of primary inoculum, which

may aggravate disease epidemics in future crops [63]. Also,

[64] reported that infusion of pea seed with the fungicide

metalaxyl before coating it with conidia of T. harzianum

improved survival of conidia in the rhizosphere compared

with the survival in the rhizosphere from seed that received

conidia only. Furthermore, [65] studied the survival of two

bacterial strains in two soils of different texture cropped

with wheat. They found that B. subtilis populations declined

rapidly in both soils and then stabilized at the levels of

added spores. P. fluorescens showed a slow, steady decline

in both soils; survival was better in the finer-textured soil, a

silt loam, than in the coarser loamy sand. For both bacteria,

some translocation to deeper soil layers was observed. No

significant rhizosphere effects were detected in either of the

two soils. Also, [66] reported that the survival of B. subtilis

NB22-1, was investigated in four different soils. After a

gradual decline, the bacterial viable cell number stabilized at

a level of 104–10

5 colony forming units/g-dry soil

irrespective of soil differences. The best survival of B.

subtilis MBI 600 occurred as spores in sterilized soil, and

spore applications to pasteurized soil in an integrated control

strategy may allow sufficient establishment of the bio-

control agent to target pathogens causing damping-off [67].

V. LONG ACTIVITY OF STORED FORMULATED

BIO-AGENTS AGAINST SOILBORNE PLANT

PATHOGENIC FUNGI

Fungal disease control is achieved through the use of

fungicides which is hazardous and toxic to both people and

domestic animals. This leads to environmental pollution.

Therefore, a more balanced, cost effective and eco-friendly

approach must be implemented and adopted by farmers.

Biological control is an innovative, cost effective and eco-

friendly approach. Use of natural enemies to control disease

is termed biological control. Biological control is an

alternative to the use of chemical pesticides. Biological

fungicides may act to suppress the population of the

pathogenic organisms through competition with pathogenic

organisms. Stimulated plant growth, which may allow plants

to quickly outgrow any pathogen effects, or damage the

pathogen by means of toxins produced [68]. Evaluation the

long term viability and activity of some stored antagonistic

fungal and bacterial agents against some root rot pathogens

was carried out in vitro[69]. They added that viability and

antagonistic ability of stored formulated bio-agents were

tested periodically throughout ten months of storage. They

found that carriers of (Sawdust) and (Sawdust + CMC) were

the most suitable tested carries for keeping the viability of B.

subtilis, P. fluorescens and T. harzianum with no significant

reduction all over the storage period up to ten months.

Meanwhile, these antagonists started to lose their viability

significantly when formulated on (Sawdust + Talc powder +

Chitosan) carrier after the third month of storage followed

by the carrier (Sawdust + Chitosan) and (Sawdust + Talc

powder) after the fifth month and finally the carrier

(Sawdust + CMC +Talc powder) after six months of storage.

The inhibitory effect of stored antagonistic fungi and

bacteria against the linear growth of root rot pathogenic

fungi was evaluated in vitro. The tested pathogenic fungi

were Sclerotium rolfsii, Rhizoctonia solani, Fusarium

solani, F. oxysporum, Sclerotinia sclerotiorum, S. minor,

Macrophomina phaseolina Pythium sp. and Alternaria

solani. No significant differences were observed in the

antagonistic ability of B. subtilis, P. fluorescens and T.

harzianum either stored on (Sawdust) and (Sawdust + CMC)

or fresh cultures against tested pathogens. Also, the

antagonistic ability of B. subtilis, the highest tested

antagonist, against tested pathogenic fungi showed lesser

reduction for losing antagonistic ability when formulated on

(Sawdust), (sawdust + CMC) carriers and stored for ten

months comparing with formulation on (Sawdust +

Chitosan), (Sawdust + Talc powder), (Sawdust + CMC

+Talc powder) and (Sawdust + Talc powder + Chitosan)

carriers and stored for the same period. Similar trend was

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also observed with the other formulated antagonists P.

fluorescens and T. harzianum. Referring to the obtained

results they conclude that the antagonistic ability not

depended on the counts or population of the antagonist but

mainly on its active viability. These findings were

confirmed with other reports. Various carriers and polymers

have been used to increase the survival rate of the organism

with mixed success. Some of the biological control agents

are adversely affected by the combination with some

traditional chemical seed protection. These products come in

dry formulations as dusts, dry spores, and gum/talc powders.

Many liquid formulations are also available for sprays, dips,

fluid drilling gels and solid matrix priming. These may be

designed for large-scale application or planter box

treatments [70]. The market for biological control products

is not only determined by agricultural aspects such as the

number of diseases controlled by one bio-control product in

different crops but also by economic aspects as cost-

effective mass production, easy registration and the

availability of competing means of control including

fungicides. Shelf life is a very important parameter to be

considered in the development of a formulation, because

most products will have to be stored for long periods of time

before they can be marketed and later applied. More recently

commercial formulations of biological controls have been

developed which have consistently given good control of

some plant diseases [71]. Also, [70] reported that the effect

of storage time at room temperature on the viability of

Trichoderma spp. in the prepared formulations showed that

more than 40% viability of the colonies was recorded at

room temperature storage after 4 months. The lowest

viability was observed in all fungal formulates after 4

months. Similar results were obtained by [72] who declared

that formulations of Trichoderma spp. after stored at

ambient conditions for 6 to 8 months. However, [73]

mentioned that no viability was observed in different soils at

30°C after 9 weeks, whereas there was viability in all soils

at 4°C even after 24 weeks. In nature wide range of organic

substrates could be used for the solid-state fermentation for

mass multiplication. Solid fermentation media consisting of

inert carriers with food bases was used for mass production

of bio-control agents [74]. The media with relatively low

microbial content would be suited for solid-state

fermentation and for the amendment of bio-control agents.

Solid substrates include straws, wheat bran, sawdust,

moistened bagasse, sorghum grains, paddy chaff, and

decomposed coir pith, farmyard manure and other substrates

rich in cellulose for inoculums production. In the present

study formulated bio-agents on base of sawdust and sawdust

+ CMC was found to be the most suitable carriers tested for

keeping both viability and antagonistic ability of stored both

fungi and bacteria bio-agents for up to ten months (300 day).

Also, Vidhyasekaran and Muthamilan, 1995 recorded that

Pseudomonas fluorescens strains showed inhibitory action

against the chickpea (Cicer arietinum) wilt pathogen

Fusarium oxysporum f. sp. ciceris under in vitro studies.

They assessed the efficacy of various carriers in sustaining

the population of these strains during storage and found that

in talc-based and peat-based formulations the bacteria

survived even up to 240 days of storage although the

population declined from 30 days, while chickpea seeds

treated with talc-based formulations, P. fluorescens survived

on the seeds for at least 180 days. Furthermore, growth

population and viability of antagonists–primed seeds during

storage were reported also by several workers. Also, it was

found [65] that T. harzianum strain T-22 increased 10-fold

during matrix priming of tomato and cucumber seeds.

Viability of the encapsulated T. harzianum remained high

for at least six months when stored at 5oC. The suppressive

ness of Zeolite- and peat-based of Paenibacillus sp. and

Streptomyces sp. formulation stored at room temperature or

at 4o

C was retained for over six months [76]. On the other

hand several carriers for formulating bio-agents had been

reported. Formulations of fluorescent Pseudomonas were

developed through liquid fermentation technology. The

fermented biomass was mixed with different carrier

materials (Talc/ Peat/ Kaolinite/ Lignite/ Vermiculite) and

stickers [77]. Also, [78] developed talc based formulation of

P. fluorescens for the management of rice blast caused by

Pyricularia grisea, in which methyl cellulose and talc was

mixed at 1: 4 ratio and blended with equal volume of

bacterial suspension at a concentration of 1010

cfu/ml. Talc

based strain mixture formulation of fluorescent

pseudomonades was developed by mixing equal volume of

individual strains and blended with talc [79]. Talc based

strain mixtures were effective against rice sheath blight and

increased plant yield under field conditions than the

application of individual strains. Talc and peat based

formulations of P. chlororaphis and B. subtilis were

prepared and used for the management of turmeric rhizome

rot [80]. One school of thought explains that CMC is added

as a sticker at 1:4 ratios to talc. Though it is effective in

disease management, it would lead to the increase in the

production cost, which would prevent the growers to adopt

the technology. More over another school of thought explain

that CMC and talc should be used at 1:100 ratios. Hence

feasibility of the technique and shelf life of the product has

to be evaluated to make the technology as a viable

component in disease management so as to promote organic

farming. So far, few biological control agents have achieved

success under field conditions. Among the hundreds of

organisms identified as potential biological disease control

agent, only few have resulted in proving commercially

acceptable control of these diseases [81]. A fungal bio-

control preparation for control or prevention of plant fungal

diseases comprises sporulated fungal biomass and a carrier

preferably is vermiculite. Different formulations have been

used in control soil-borne pathogens, these are, fungal

spores [82], and powdery preparations of fungal mycelium

[83]. A bio-control formulation with agricultural potential

should possess several desirable characteristics such as: easy

preparation and application, stability, adequate shelf life,

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46

abundant viable propagules, and low cost [84]. The

formulation should be amenable for application to both

phylloplane and rhizosphere, depending on the pathogens

and plants to be controlled. Formulation of the bio-agents to

reduce incidence of the diseases caused by soil-borne

pathogens in the field is of great importance in bio-control

of such diseases. Therefore the reported work by [69] was

aimed also to determine the efficacy of application of

formulation contained the B. subtilis, P. fluorescens and T.

harzianum as seed treatment against root rot diseases of

Cucumber and Pepper under greenhouse conditions. In this

study, the efficacy of using stored formulated various

antagonistic fungi and bacteria as seed treatment against root

rot incidence of cucumber and pepper was evaluated in pots

experiment using soil artificially infested with the disease

incidents under greenhouse conditions. They added that all

the tested fresh and ten months stored bio-agents showed

interesting highly significant effect causing high reduction

of root rot incidence at both pre-, and post-emergence stages

of plant growth comparing with the check treatment. Their

results revealed that all the tested fresh and stored bio-agents

showed interesting highly significant effect causing high

reduction of root rot incidence at both pre-, and post-

emergence stages of plant growth comparing with the check

treatment. No significant differences were observed between

fresh applied bio-agents cultures and stored ones formulated

on (Sawdust) and (Sawdust + CMC) carriers. Meanwhile

stored bio-agents on (Sawdust + Chitosan); (Sawdust +Talc

powder); (Sawdust + CMC +Talc powder) and (Sawdust

+Talc powder + Chitosan) carriers showed less significant

protective effect against root rot disease incidence. They

showed low significant pre-emergence root rot incidence

ranged between 23.3-36.6% for applied fungal and bacterial,

respectively comparing with untreated and check treatments

which recorded as 54.6 and 51.9% for cucumber and pepper,

in respective order. Similar trend concerning post-

emergence root rot incidence was observed. All treatments

varied in their effect on disease incidence. Treated seeds

showed higher significant reduction on disease incidence

than untreated ones. Moreover, bacterial treatment showed

superior effect on disease incidence (11.5-14.2%) followed

by fungal treatment (14.2-20.0%). Also, [69] reported that it

is also interesting to note that, more reduction in disease

incidence was observed at post-emergence stage of plant

growth than at pre-emergence. This observation could be

attributed to their sensitivity to the fluctuations in

environmental conditions and are inconsistent in their

performance. Similar observation was also reported by

several investigators. It was recorded that [85] 60-75% of

the cotton crop in US is treated with B. subtilis for the

management of soil borne pathogens encountered in cotton

ecosystem. Among several PGPR strains Bacillus based

products gains momentum for commercialization. Because,

Bacillus spp., produce end spores tolerant to extremes of a

biotic environments such as temperature, pH, pesticides and

fertilizers [85]. Furthermore, seed treatment of pigeon pea

with talc based formulation of fluorescent pseudomonads at

the rate of 4g/kg of seed followed by soil application at the

rate of 2.5 kg/ha at 0, 30, and 60 days after sowing

controlled pigeon pea wilt incidence under field conditions.

The additional soil application of talc based formulation

improved disease control and increased yield compared to

seed treatment alone [86]. Delivering of P. fluorescens as

seed treatment followed by three foliar applications

suppressed rice blast under field conditions [78]. Combined

application of talc based formulation of fluorescent

pseudomonads comprising of Pf1 and FP7 through seed

treatment, seedling dip, soil application and foliar spray

suppressed rice sheath blight and increased plant growth

better than application of the same strain mixture either

through seed, seedling dip or soil (Nandakumar et al., 2001).

Biological control of plant pathogens is becoming an

important component of plant disease management

practices. In case study of [69] the used fungal and bacterial

antagonists proved their highly inhibitor effect against root

rot pathogens under in vitro and in vivo conditions. These

results are also confirmed by several researchers [11,58,59].

VI. DIFFERENT APPROACHES OF INTRODUCING

BIO-AGENTS TO THE SOIL FOR CONTROLLING

ROOT ROT PATHOGENS

Root rot in vegetables strikes quickly and then ruin a

whole crop. However the largest instruction course of

actions is preventative measures, as therapy with fungicide

does not normally work. Biological control of seedling

diseases using antagonistic fungi and bacteria has received

increasing attention. Antagonists applied to seeds prior to

planting colonize the rhizosphere of seedlings and thus are

present at or near the pathogen’ infection court, where they

act by producing antifungal or antibiotic compounds,

through hyper parasitism, or by competitively colonizing

sperm sphere and rhizosphere substrates [60]. (Application

of biological control using antagonistic microorganisms had

been recorded by several investigators. In this concern, [87]

evaluated different approaches of some antagonistic fungal,

bacterial and yeast agents applied as seed treatment or soil

drench against various soil-borne pathogens causing

vegetables (cucumber, cantaloupe, tomato and Pepper) root

rot disease under greenhouse conditions. In this study, the

tested pathogenic fungi were Alternaria solani Fusarium

solani, F. oxysporum, Rhizoctonia solani, Sclerotium rolfsii,

Macrophomina phaseolina and Pythium sp., meanwhile the

tested bio-agents were Trichoderma harzianum, T. Viride

and Bacillus subtilis, Pseudomonas fluorescens and

Sacchromyces serevisiae. They reported that the applied bio-

gent treatments either as seed soaking or soil drench caused

a significant effect on root rot incidence at both plant growth

stages of grown vegetables comparing with control.

Regarding root rot incidence at pre-emergence stage, a

significant effect was observed in bio-agents treatment as

seed soaking comparing with soil drench treatment. The

treated seeds showed a protective effect for seeds

germination against the invasion by soil-borne pathogenic

fungi. Their data also revealed that the antagonist T.

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47

harzianum showed significant superior effect to reduce

diseases incidence followed by B. subtilis. Also, the

antagonists T. viride and P. fluorescence occupied

significantly the second degree for reducing root rot

incidence. The treatment with S. serevisiae had the lowest

effect on disease incidence, although it significantly lesser

than check control treatment. This observation was true with

all vegetables tested. In this regards, the antagonistic fungi

were more actively than the two antagonistic bacteria for

reducing root rot incidence at pre-emergence stage of plant

growth. Furthermore, data also showed that the lowest root

rot incidence was recorded with Cantaloupe and cucumber

followed by Tomato and Pepper plants. Data also revealed

that the pathogenic fungi S. rolfsii, R. solani, F. solani, F.

oxysporum, Pythium sp., M. phaseolina and S. sclerotiorum

showed more response to antagonistic fungi and bacteria

which reflected in recorded minimization of the root rot

incidence. They added that soil drenched with different bio-

agents showed more efficient for reducing root rot incidence

at post-emergence growth stage of tested vegetables. In this

concern, the obtained data revealed that percentage of root

rot incidence highly reduced in soil drenched with T.

harzianum comparing with seed soaking treatment with the

same fungus for tested vegetables, Cucumber, Cantaloupe,

Tomato and Pepper. Similar observations were also

recorded in soil drench treatment with T. viride, B. subtilis,

P. fluorescens and S. serevisiae. Furthermore, T. harzianum

and B. subtilis drenched soil showed interested results

against root rot pathogens that complete reduction (100%) in

disease incidence was recorded for Cucumber and

Cantaloupe in infested soil with the most tested pathogenic

fungi comparing with 90-89.1% infection recorded in

control check treatment. The case study of [87] showed that

bio-agents applied as soil drench was the most favorite

method of application resulted in the best control records.

They conclude that the obtained reduction in invaded

vegetable plants with root rot pathogens may be attributed to

the high accumulative inoculum potential of the introduced

bio-agents into the root region, before sowing and

throughout the growing season as well, where they are

predicted to have a direct impact on already established

pathogens population. Similar explanation was reported by

[11], who stated that, soil treatment with biocide T.

harzianum showed better reduction in root rot incidence of

bean followed by seed coating with the biocide. He added

that, these differences could be due to the initial inoculum of

T. harzianum introduced into the soil. Moreover, the high

fungal population density introduced through soil treatment

technique enables the fungus to adapt itself against

environmental conditions [64] resulting in dominance of

high population of the introduced fungi in the plant

rhizosphere. The use of microorganisms that antagonize

plant pathogens (biological control) is risk-free when it

results in enhancement of resident antagonists. Moreover,

the combination of such biological control agents (BCAs)

with reduced levels of fungicide (integrated control)

promotes a degree of disease suppression similar to that

achieved with full fungicide treatment [89]. Moreover, the

application of biological controls using antagonistic

microorganisms has proved to be successful for controlling

various plant diseases in many countries [10,59,61,62,90].

Furthermore, for the effective biological control of soil-

borne plant pathogens, a major consideration has been given

to proliferation of the antagonist after introduction into the

soil. Among the desirable attributes of a successful

antagonist is its ability to produce inoculum in excess and to

survive, grow, and proliferate in soil and the rhizosphere

[90]. Various actinomycetes, bacteria, and fungi, which

show antagonism to P. capsici, exist in soils where peppers

are grown [91,92,93]. In particular, some antagonistic

rhizobacteria such as Burkholderia cepacia [92] and

Pseudomonas aeruginosa [93] were very effective against

Phytophthora blight in pepper plants under laboratory and

greenhouse conditions. Similarly, application of B. cepacia

granules into soil provided better suppression of

Phytophthora blight on red-pepper seedlings, as compared to

direct drenching with Burkholderia cepacia suspensions

[94]. Soil drenches and dipping of seedling roots with the

antagonist suspensions were found to be more effective in

disease suppression than the coating and dipping pepper

seeds [92]. Also, [95] reported that Trichoderma are present

in all soil and they are the most cultural fungi. Trichoderma

species are strongly antagonistic to other phytopathogenic

fungi. They produce hydrolytic enzymes which are believed

to play an important role in the parasitism of

phytopathogenic fungi. The study of [87] demonstrate that

the use of bio-agent treatments, B. subtilis, P. fluorescens

and S. serevisiae either as seed soaking or soil drench could

reduce root rot incidence of tested vegetables grown in soil

artificially infested with disease incidents. These results are

in agreements with previous reports in this concern [59,96].

Several workers explained the mode of action of

antagonistic bacterial and yeast isolates. In this regards, the

potential of Bacillus sp. to synthesize a wide variety of

metabolites with antifungal activity is known and in recent

years it has been a subject of experiments [97]. Most of

these substances belong to lipopeptides, especially from

surfactin, iturin and fengicin classes. Not so much is known

about the mechanism of antifungal activity of these

substances produced by Bacillus sp. Some of them (iturin

and surfactin) are able to modify bacterial surface

hydrophobicity and, consequently, microbial adhesion to

surfaces (to mycelium) [97]. Antibiotics of the iturin group

were found to act upon the sterol present in the cytoplasmic

membrane of the fungi [98]. Biological control of

Aspergillus niger by Bacillus subtilis was also investigated

by [99]. They demonstrated that the bacterial cells initially

adhered to the fungus, multiplied and extensively colonized

the surface. Rapid growth of bacterial cells on the surface

resulted in damage of fungal cell walls. These aspects

appear essential in association with the antifungal properties

of Bacillus sp. used in the biological control of plant

diseases. Moreover, although the biocontrol activity of

antagonistic bacteria and yeasts has been demonstrated on a

variety of commodities, the mode of action of these

microbial bio-control agents has not been fully elucidated.

In the case of bacterial antagonists, it has been suggested

that their bio-control activity may be partly associated with

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48

the production of antibiotics [100,101]. Antagonistic yeasts

have been selected mainly for their capability to rapidly

colonize and grow in surface wounds, and subsequently to

compete the pathogen for nutrients and space. Competition

for nutrients and space is believed to be the major

component of the mode of action of antagonistic yeasts

[102,103]. Besides competing for nutrients and space,

antagonistic yeasts parasitize major post harvest pathogens

directly, through strong attachment to their hyphae. This

leads to partial disruption of hyphal wall structures [103].

Seed treatment is an attractive delivery system either fungal

or bacterial bioprotectants [61]. Bio-protectants applied to

seeds may not only protect seeds [12] but also may colonize

and protect roots [62]. The term bio-priming has been used

by at least research groups, and each group has used a

different technique to achieve bio-priming. In addition, bio-

control agents were added directly to the Solid Matrix

Priming (SMP), which allowed the T. harzianum to colonize

the seeds during the priming process [82]. On the other

hand, [20] added a suspension of the bacterium

Pseudomonas fluorescens to 1.5% methyl cellulose coated,

surface sterilized sweet corn seed prior to hydrating the

seeds between moistened paper towels. The two methods

were used to protect tomato and sweet corn against Pythium

damping-off. In the present study bio-priming cucumber and

pepper seeds bio-treatment was applied through mixing with

stored formulated bio-agents or imbibing seeds into solution

of fresh bio-agents cultures for 1h as a period time for

allowing seeds’ colonization before drying. A successful

antagonist should colonize rhizosphere during seed

germination [104]. It is evident that antagonistic bio-agent

can affect plant resistance to a pathogen either by inducing

the basal level of defense reactions immediately after

treatment or by enhancing a capacity for rapid and effective

activation of cellular defense responses, which are induced

only after contact with a challenging pathogen, a process

known as “sensitization” or “priming” [105]. Also, [106]

reported that based on microscopic evaluation of the growth

a distribution of the antagonist during priming,

Clonostachys rosea colonized the whole surface of the

pericarp, including the apex of carrot seed where the

primary root emerges. These reports are in agreements with

the obtained results recorded by [69].

VII. PLANT RESISTANCEINDUCERSAND/OR BIO-

AGENTS APPROACHES AGAINST ROOT DISEASES

INCIDENCE

The objective of the reviewed work recorded by [107]

was to evaluate the suppression activity of some fungicides

alternatives applied as soil drench before transplanting

against root rot incidence of some vegetables under

commercial plastic houses conditions. This research

focused on finding compounds that are safe to humans and

the environment, e.g. chemical resistance inducers and

essential oils combined with bio-agents. The obtained

results [107] showed the root rot incidence of Cucumber

seedlings grown in plastic house at Dokki and Tookh

locations, Giza and Qualiobia governorates. These data

revealed that all applied treatments have significant drastic

effect on root rot incidence comparing with untreated

control. In addition, the highest reduction in root rot

incidence of cucumber plant either grown at Dokki or

Tookh locations was observed at the applied treatments of

the bio-agent [Trichoderma harzianum]; a mixture of:

[Humic & Folic acids +Furfural +Thyme oil]; a mixture of:

[Furfural +Trichoderma harzianum]. Meanwhile, the lowest

reduction in root rot incidence was recorded at the bio-agent

Pseudomonas fluorescens treatment for cucumber plant

grown at Dokki and Tookh locations, respectively.

Similarly, data recorded by [107] revealed that the applied

treatments of a mixture of: [Furfural +Trichoderma

harzianum] followed by a mixture of: [Humic & Folic acids

+Furfural +Thyme oil] and the bio-agent [Trichoderma

harzianum], caused the highest reduction in root rot

incidence of tomato plants grown at Haram location. As for

Pepper plants, the highest reduction in root rot incidence

was recorded at the applied treatments, a mixture of:

[Humic & Folic acids +Furfural +Thyme oil]; a mixture of:

[Furfural +Trichoderma harzianum] and mixture of:

[Humic & Folic acids + Bacillus subtilis], respectively. The

lowest reduction in root rot incidence was recorded at the

bio-agent Pseudomonas fluorescens treatment for Tomato

and pepper plants, respectively. In case study of [107] the

obtained results showed high efficacy of the bio-agents

application alone or combined with the fungicides

alternatives Humic & Folic acid, Furfural or Thyme oil as

soil drench treatment before transplanting. A mixture of:

[Humic & Folic acids + Furfural +Thyme oil] and the bio-

agent [Trichoderma harzianum] treatments could

completely inhibit the root rot disease incidence of

cucumber plants grown at Dokki location and also reduced

the disease incidence by 90.4 and 92.2% at Tookh location.

Also, the highest reduction in root rot incidence of both

tomato and pepper was also recorded at treatment of the

bio-agent [Trichoderma harzianum] alone or combination

treatments of [Furfural +Trichoderma harzianum] and

[Humic & Folic acids + Furfural +Thyme oil]. They

conclude that the obtained reduction in invaded vegetable

plants with root rot pathogens may be attributed to the high

accumulative inoculum potential of the introduced bio-

agents into the root region, before sowing and throughout

the growing season as well, where they are predicted to

have a direct impact on already established pathogens

population. In this regards, plant products are characterized

as having a wide range of volatile compounds could be used

as alternative anti-bacterial and anti-fungal treatments [25].

It is evident from reviews by several investigators that

Humic and Fulvic acids have been early recorded to have

appositive effect against plant pathogens and their cells

biological activities [26,27,28]. On the other hand, furfural

is a naturally occurring compound, and recently used as a

new pesticide active ingredient intended for the use as a

fumigant to control root infesting plant parasitic nematodes

and fungal plant diseases. Moreover, [29] reported that most

of drip irrigation treatments reduced populations of Pythium

ultimum and F. oxysporum and increased stem height

compared with the nontreated controls. Metham sodium,

furfural + metham sodium, sodium azide, and chloropicrin

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49

significantly reduced the incidence of Liatris stem rot

caused by Sclerotinia sclerotiorum. Similar explanation was

also reported by [11] who stated that, soil treatment with

biocide T. harzianum showed better reduction in root rot

incidence of bean followed by seed coating with the

biocide. He added that, these differences could be due to the

initial inoculum of T. harzianum introduced into the soil.

Moreover, the high fungal population density introduced

through soil treatment technique enables the fungus to adapt

itself against environmental conditions [64] resulting in

dominance of high population of the introduced fungi in the

plant rhizosphere. The use of microorganisms that

antagonize plant pathogens (biological control) is risk-free

when it results in enhancement of resident antagonists.

Moreover, the combination of such biological control

agents (BCAs) with reduced levels of fungicide (integrated

control) promotes a degree of disease suppression similar to

that achieved with full fungicide treatment [88].

Furthermore, for the effective biological control of soil-

borne plant pathogens, a major consideration has been

given to proliferation of the antagonist after introduction

into the soil. On the other hand, a more balanced, cost

effective and eco-friendly approach must be implemented

and adopted farmers. In order to overcome such hazardous

control strategies, scientists, researchers from all over the

world paid more attention towards the development of

alternative methods which are, by definition, safe in the

environment, non-toxic to humans and animals and are

rapidly biodegradable. Such strategy is use of fungicides

alternatives, i.e. plant resistance inducers

[108,109,110,111]. or essential oils [112,113]. Humic acid

(HA) is a heterogeneous mixture of many compounds with

generally similar chemical properties it performs various

functions in the soil and on plant growth. Humic substances

have been early recorded to have appositive effect against

plant pathogens [26,28]. Also, many studies [26,114]

showed that Fulvic acid (FA) have a greater effect on cells

biological activities than humic acids (HA) compounds. The

addition of 500 mgl_1

of humic acids on the growth medium

completely eliminated the inhibition of P. ultimum by R.

radiobacter [115]. Furthermore, in vitro, humic acid at

15.0% (v/v) reduced significantly the radical growth and

spore germination of Fusarium solani the causal agent of

dry root rot [116]. Moreover, recently, plant products with

antimicrobial properties have notably obtained attention as

possible applicants in order to prevent bacterial and fungal

growth [117]. Plant products are characterized as having a

wide range of volatile compounds. This means that essential

oils could be used as alternative anti-bacterial and anti-

fungal treatments [25]. It is evident from reviews by

[118,119] some plant extracts and essential oils exhibited

antifungal properties. Also, [120] showed that essential oil

of Juniperus communis may be applicable against a range

of damping-off diseases. However, not much can be found

in the literature regarding the efficacy of furfural against

fungi and bacteria, the metabolism and effects of furfural in

eukaryotic cells have been investigated for yeast cells. In

this case, the conversion of furfural depends on the rate of

oxidizing in yeasts. Furfural is oxidized to fluoric acid

under aerobic conditions, and it is reduced to furfuryl

alcohol in anaerobic fermentation [121]. The authors

indicated that when furfural was added to the culture

medium, both cellulose and β-glucosidase activities

decreased with increasing furfural concentration. The

activity of both enzymes decreased by 50% when

concentration of furfural increased from 0 to 1.2 g/l (1200

ppm). Furthermore, [122] demonstrated that soil treatments

with furfural control southern blight caused by S. rolfsii in

lentil, while stimulating development of Trichoderma spp.

and bacteria antagonistic to S. rolfsii. There are a few cited

reports explaining the furfural mode of action against soil

micro flora. In this regard, the end-use product containing

90% furfural in a liquid formulation is registered as

commercial products, e.g. Crop guard, Multigaurd protect

and Protect etc. (Anonymous 2005, 2006). Pamphlet sheet

of Protect (2005–2006) has demonstrated efficacy in the

control of plant parasitic nematodes and fungal pathogens,

i.e. Pythium, Fusarium, Phytophthora and Rhizoctonia.

Protect is a contact soil treatment that kills nematodes by

irreversibly damaging the cuticle and kills fungi by reacting

with the cellular wall and disrupting cellular functions.

Also, it is obvious from Multigaurd fate sheet that it

controls root infesting fungal plant pathogens such as

Pythium, Phytophthora, Fusarium and Rhizoctonia. Also,

[123] reported that under in vitro conditions, the linear

growth of tested soil borne pathogenic fungi was

dramatically reduced with the increasing of furfural

concentrations added to the growth medium up to 4000 ppm

where no growth was observed, while the bacterial and

fungal bio-agents showed more tolerance to these

concentrations and failed to grow at 6000 and 7000 ppm,

respectively. They added that pot and field experiments

indicated that furfural at 6000 ppm combined with bio-

agent treatments proved to have superior suppressive effect

against tomato root rot incidence, caused by Fusarium

solani and Rhizoctonia solani, comparing with each

individual treatment. All these reports confirm the findings

reported by [107].

VIII. CONCLUSION

Because plant population densities in greenhouses are

usually very high and closely confined by the greenhouse

walls, some virus diseases, foliar blights, leaf spots, stem

and fruit rots, root rots and other diseases can become severe

very quickly. Use strict sanitation procedures for

germinating seed and growing transplants are needed. These

procedures are recommended for all greenhouses vegetable

and herb growers to minimize the risk of introducing plant

pathogens and reduce disease severity if pathogens are

present. Promising applicable technique could be suggested

on the light of the results stated in the present review. The

usage stored formulated bio-agents might be considered as

safe, cheep and easily applied bio-control method as seed or

soil application against such soil-borne plant pathogens

particularly in organic farms taken in consideration

avoidance of environmental pollution. Also, the obtained

results in the present review appears that an urgent

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50

investigation of favorable possible applicable method for

controlling root diseases, where the addition of a biological

control agent in combination with plant resistance inducers,

or essential oils could be resulted in increased symptomless

plant stand. These methods characterized as environmentally

safe, bioactive natural products which able successfully to

control phytopathogenic fungi in crop production systems.

ACKNOWLEDGEMENT

This work was supported financially by the Science and

Technology Development Fund (STDF), Egypt, Grant No.

1059.

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AUTHOR BIOGRAPHY First Author Department of Plant Pathology, National Research Centre – Egypt, Email: [email protected]

Second Author: Department of Plant Pathology, National Research Centre – Egypt, Email: [email protected].

Third Author: Department of Vegetable Research, National Research Centre –Egypt, Email: [email protected]

mailto:[email protected]



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