Biological Soil Treatment With Trichoderma Harzianum To Control Root Rot

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Biological soil treatment with Trichoderma harzianum to control root rot

disease of grapevine (Vitis vinifera L.) in newly reclaimed lands in Nobaria
province
Article in Archives of Phytopathology and Plant Protection · January 2010

DOI: 10.1080/03235400701722004
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disease of grapevine (Vitis vinifera L.) in newly reclaimed lands in Nobaria
province
Riad S. R. El-Mohamedy a; E. H. Ziedan a;A. M. Abdalla a
a
Plant Pathology Department, National Research Centre, Giza, Egypt
Online publication date: 09 December 2009
To cite this Article El-Mohamedy, Riad S. R. , Ziedan, E. H. andAbdalla, A. M.(2010) 'Biological soil treatment with
Trichoderma harzianum to control root rot disease of grapevine (Vitis vinifera L.) in newly reclaimed lands in Nobaria
province', Archives Of Phytopathology And Plant Protection, 43: 1, 73 — 87
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Archives of Phytopathology and Plant Protection
Vol. 43, No. 1, 1 January 2010, 73–87

disease of grapevine (Vitis vinifera L.) in newly reclaimed lands in
Nobaria province
Riad S.R. El-Mohamedy*, E.H. Ziedan and A.M. Abdalla
Plant Pathology Department, National Research Centre, Giza, Egypt

(Received 16 August 2007)
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Augmentation of soil with different biological treatments i.e., Trichoderma

harzianum cultured on sugar cane bagasse, Trichoderma harzianum (spore
suspension 5 6 106 cfu/ml) and plant guard (biocide), successfully controlled
Fusarium solani, F. oxysporium and Macrophomina phaseolina, the main
pathogens of root rot disease on grapevines in Nobaria province. Complete
reduction of these pathogens was recorded at 4 ml/l of plant Guard. Meanwhile,
T. harzianum caused a reduction of 80.0, 84.4 and 88.9% of linear growth of the
same pathogens respectively. In greenhouse trials, artificially infested soil treated
with bio-enhancing bagasse at rate of 10% (w/w) of soil, plant guard 4 g/l and T.
harzianum 100 ml/l treatments showed a large effect in decreasing percentages of
infection and severity of root rot disease on grapevine seedlings. These treatments
caused a reduction in root rot infection caused by M. phaseolina, F. oxysporum
and F. solani reach 100, 100, 80% and 80, 60, 60% and 60, 40, 40%, respectively,
with two soil applications. Also, the percentage reduction in disease severity reach
100, 100 and 88.2%; 69.2, 57.1 and 52.9%; 46.2, 42.8 and 41.2% respectively.
Meanwhile, one soil application with the same treatments caused a moderate
effect in decreasing both disease infection and severity. Two soil applications
with bio-enhancing bagasse 10% and plant Guard 4 ml/l treatments caused
significantly reduced numbers of infested vines as well as percentage of disease
severity of these vines after 30, 60 and 120 days from application. Moreover, these
treatments cause an increasing population density of Trichoderma spp., wherever
it decreases in the rhizosphere soil of untreated vines (control). The large increase
in yield/vine was recorded on vines treated with bio-enhancing bagasse 10%
(51.2% increase over control) and Plant Guard 4 ml/l (48.8% increase over
control) in two applications. Through these results it can be suggested that
manipulating soil by biocontrol agent formulation on agricultural wastes or as
biocides such as plant guard may be safely used commercially as a substitute for
traditional fungicidal treatments for controlling soil borne plant pathogens.
Keywords: grape vine; root-rot; biological control; biocide; Trichoderma
harzianum
Introduction
Grape (Vitis vinifere L.) is one of the most distributed orchards in the world. Grapes
are the most favorable fruit crop grown in newly reclaimed lands in the desert in
*Corresponding author. Email: riadelmohamedy@yahoo.com
ISSN 0323-5408 print/ISSN 1477-2906 online

Ó 2010 Taylor & Francis
DOI: 10.1080/03235400701722004
http://www.informaworld.com
74 R.S.R. El-Mohamedy et al.
Egypt. Meanwhile, over the few past years, root rot diseases of grapevine have
been found to be spread out over several governorates. Such diseases cause a
decline in vigor and yield of vineyards. Root rot disease of grapevine was attributed
to many soil borne plant pathogens such as Rhizoctonia solani (Walker 1992, 1994),
Pythium ultimum (Ulkhede 1992), Phytophthora spp. (Latorre et al. 1997),
Phymatotrichium omnirorum (Ramirez-Arredondo 1994), and Fusarium wilt caused
by Fusarium oxysporium f. sp herbemantis (Andrade et al. 1995; Highett and Nair
1995).
In Egypt, Rhizoctonia solani, Fusarium solani, Fusarium spp., Fusarium
oxysporium, Pythium ultimum, Macrophomina phaseolinae and Botryiodiplodia
thiobromea were reported to be pathogenic fungi causing root rot and wilt diseases
on grapevine (Badawy 1971; Mourad 1983; Mahrous 1994; Ziedan 2003).
An investigation of controlling root rot pathogens of grapevine using biological
soil treatments as a substitute for chemical fungicides is considered very important,

particularly in newly reclaimed sandy soil, where grapevine is an important crop.
Application of biological control trails using antagonistic microorganisms has
proved to be successful for controlling various soil borne plant pathogens (Yehia
et al. 1994; Abdel-Kader 1997; Prasad and Ragashwaran 1999; Reuter and Kubiak
2003; Weakert-Whitelaw 2004; El-Mohamedy 2004; El-Mohamedy et al. 2006).
Nemec et al. (1996) noted that amended planting mixes with formulations of
commercial biocontrol agent such as T. harzianum, Bacillus subtilis, Gilocladium
virens, and Streptomyces spp. reduced root rot and grown rot disease on tomato, bell
pepper, celery and citrus.
The purpose of this research is to evaluate the efficiency of different forms of
T. harzianum as biological soil treatments in controlling root rot pathogens of
grapevine under greenhouse and field conditions.
Materials and methods

Causal organisms
Pure cultures of Fusarium oxysporium, Schlecht, Fusarium solani (Mart.) Sacc. and
Macrophomina phasealina (Tassi) Goid were previously isolated from roots of
grapevines affected by root rot disease. Such isolates have been recorded to induce
root rot and wilt on grapevine seedlings during previously studies (Ziedan 2003;
Ziedan et al. 2005).
Biocontrol agent and biocides

Trichoderma harzianum NB isolate was previously isolated and used as a biocontrol
agent against many soil borne pathogens (El-Mohamedy 2004; El-Mohamedy et al.
2006). Plant guard is the commercial biocide (original compound is T. harzianum
3 6 107 spore/ml) broached from El-Nasser Co. Egypt.
Effect of T. harzianum and plant guard on root rot pathogens in vitro

The antagonistic ability of T. harzianum against F. oxysporium, F. solani and
M. phaseolina was assessed on PDA medium using the dual culture technique
(Ferreria 1991). The inhibitory effect of plant guard on the same pathogens was
tested as the following.
Archives of Phytopathology and Plant Protection 75
Three doses of plant guard i.e., 1, 2 and 4 ml/l were prepared in PDA medium.
Petri dishes were inoculated at the centre with 5 mm discs of seven days old each
pathogen culture. Control treatment was PDA medium only. Five replicates were
used for each particular treatment as well as control. All Petri dishes were incubated
at 258C for eight days then examined. The percentage growth reduction of each
pathogen was calculated in relation to the growth in control treatment.
Inoculate preparation
Pathogenic fungi
Fungal inoculums of each F. oxysporium, F. solani and M. phaseolinae were prepared
a according to Tsao and Garber (1960). Spore suspension of T. harzianum was
grown on PDA medium in a one liter conical flask for seven days at 258C. Spore
suspension was prepared by adding sterile deionized water to the cultures and
scraping the agar surface with a flamed glass rod. The resulting suspension was
adjusted to a concentration of 5 6 106 spore/ml.
Bio-enhancing and enhancing bagasse

The inoculums of bio enhancing bagasse (T. harzianum formulated on sugar cane
bagasse) as well as enhanced bagasse were prepared according to El-Mohamedy
(2004).
Greenhouse experiments
These experiments were carried out to evaluate the efficiency of different biological
soil treatments, i.e., drenching soil with spore suspensions of T. harzianum
5 6 106 spore/ml (50 ml/l and 100 ml/l), water suspension of plant guard (2 ml/l
and 4 ml/l), and soil treated with bio enhancing bagasse (5 and 10% w:w of soil), on
controlling root rot pathogens of grapevine under artificially infested soil in
greenhouse.
Seedlings of grapevine cv. Thompson (one year old) were transplanted in plastic
pots (20 cm diameter) containing infested sand:loam soil (2:1) with inoculum of each
pathogen individually. Five pots were used as replicates for each particular
treatment. The following treatments were evaluated:
– Bio enhancing bagasse: 50 and 100 g/kg soil.

– Enhancing bagasse: 50 and 100 g/kg soil.
– T. harzianum: spore suspension 50 and 100 ml/l.
– Plant guard (biocide): 2 ml/l and 4 ml/l.
– Control (untreated infested soil).
All soil treatments were applied at the replanting time of grapevine seedlings as
one application and 30 days later as the second application. After four months of
applying soil treatments, the number of infected seedlings was recorded and
development of root rot symptoms (severity) was determined on the scale rating
from 0 to 4 degrees where 0 ¼ health plant, 1 ¼ yellowish þ 1/3 plant wilted, 2 ¼
2/3 plant wilted, 3 ¼ whole plant wilted, 4 ¼ plant dead, according to Ziedan
(2003). Percentages of root-rot infection and severity were recorded and calculated.
Field experiment
Effect of biological soil treatments on control of root rot disease
The experiment was carried out at E1-Nobaria region, Beheira Governorate during
2004 and 2005 seasons in grapevine orchards of eight-year old cv. Thompsom
seedless, with a history of root rot pathogens of grapevine.
Eighty-one diseased grapevine trees with different degrees of disease severity i.e.
1, 2 and 3 degrees were detected during a survey done by the authors during the 2004
season. Nine diseased trees with 1, 2 and 3 degrees of disease severity were used as
replicates for each treatment as well as control.
The following soil treatments were applied.
(1) Bio-enhancing bagasse at the rate of 0.5 and 1.0 kg/vine

(2) Plant Guard at the rate of 2 and 4 ml/l

(3) Control (untreated diseased trees).
All soil treatments were applied to soil around the mean stem of vine
twice per season, first in November 2004 and the second in February 2005. After
30, 60 and 120 day from application, the number of recovered grapevines and
the degree of disease seventy on these vines were recorded and calculated as
before.
Effect of soil treatments on the colonization of pathogens in grapevine roots and

population density of T. harzianum in rhizosphere soil
Samples of feeder roots and rhizosphere soil were collected 0, 30, 60 and 120 days
after applying soil treatments. Four standardized approx. 500 g soil samples
containing feeder roots were collected at depths of 15–30 cm on the north, south,
east and west sides of the tree. Four samples from each tree in every treatment were
pooled and mixed thoroughly.
Randomly 100 selected feeder roots tips for each treatments were cut
into segments (5 mm long) sterilized for 2 min. with 5% NaOCl2, rinsed in sterile
distilled water. Ten roots segments were plated on PCNB medium (Nash and
Snyder 1962) for recovering Fusarium spp. and PDA medium for recovering M.
phasealina. Five plates were used as replicates. Plates were incubated at 258C for
seven days. The number of root segments yielding Fusarium spp. or Macrophomina
phasealinae were counted and the percentages of infected root segments were
calculated.
The method of plate count technique (Allen 1961) using PDA medium supple-
mented with 250 ppm chloromycetin (Papavizes and Lumsden 1982) was used to
determine total count of (cfu/g dry soil) of Trichoderma spp. in the rhizosphere soil
of treated and non-treated grape vine trees. Five plates were used as replicates for
each treatment. Total counts of Trichoderma spp. were expressed as cell forming
units (cfu) per gram dry soil.
Effect of biological soil treatments on grapevine yield

At the end of the season all clusters of each vine of each treatment were picked up.
The average number of clusters per vine as well as cluster weight and weight of 50
berries for each treatment were calculated.
Statistical analysis
The Tukey test for multiple comparison among means was utilized (Neler et al. 1985).
Results
Effect of T. harzianum and plant guard on root rot pathogens in vitro
Linear growth of F. oxyspirum, F. solani and M. phaseolina was affected significantly
by T. harzianum and Plant Guard (biocide); these results are given in Table 1.
Results in Table 1 indicate that complete reduction of pathogenic fungi growth
was recorded at 4 ml/l of plant Guard. However, 2 ml/l caused 80.0, 83.3 and 88.9%
reduction for F. oxysporium, F. solani and M. phaseolina, respectively. But at 1 ml/l
the smallest effect on all tested pathogens was recorded. T. harzianum caused a
reduction of linear growth of F. oxysporium, F solani and M. phaseolina by 80.0, 84.4
and 88.9%, respectively.
Effect of different soil treatments on root rot disease infection of grapevine cv.
Thompson seedlings transplanted in artificially infested soil in greenhouse
The efficiency of different biological soil treatments in controlling root rot pathogens
of grapevine under artificially infested soil was studied.
Results in Table 2 clearly show that the highly effective treatments for decreasing
percentages of root rot incidence were bio-enhancing bagasse 10% and plant guard
4 g/l followed by T. harzianum 100 ml/l. These treatments caused a reduction of root
rot on grapevine caused by M. phaseolina, F. oxysporum and F. solani reaching 100,
100, 80% and 80, 60, 60% and 60, 40, 40%, respectively with two soil applications.
Meanwhile, with one soil application with the same treatments, a moderate effect on
reducing root rot infection was recorded. The least effective soil treatments for
controlling root rot pathogens of grapevine were enhancing bagasse (5 and 10%) and
T. harzianum (50 ml/l) when they were used as a single soil application. The least
effective treatments were enhanced bagasse 5% or 10%, T. harzianum 50 ml/l and
plant guard 2 ml/l with one soil application.
Effect of different soil treatments on root rot disease severity on grapevine cv.
Thompson seedlings transplanted into artificially infested soil in greenhouse
M. phaseolina and F. oxysporium were highly affected by all biological treatments
compared with F. solani; the lowest percentages of root rot reduction were recoded
with these pathogens.
Table 1. Linear growth reduction of grapevine root rot pathogens as affected by T.

harzianum and plant guard on PDA medium in vitro.
Plant guard
T. harzianum 1 ml/l 2 ml/l 4 ml/l Control
Pathogenic fungi L* R** L R L R L R L R
F. oxysporium 18c 80 38b 57.8 18c 80 0d 100 90a 0
F. solani 14c 84.4 33b 63.3 15c 83.3 0d 100 90a 0
M. phaseolina 10c 88.9 28b 68.9 10c 88.9 0d 100 90a 0
*L: Linear growth (mm). **R: % Reduction.

Figure with the same letter in the same row are not significant at (P ¼ 0.05).
78
Table 2. Effect of different soil treatments on root rot disease infection of grapevine cv. Thompson seedlings transplanted in artificially infested soil in
greenhouse.
% root rot disease infection.

Soil treatment No. of soil application F. solani Reduction % F. oxysporium Reduction % M. phaseolinae Reduction %
Bio-enhancing bagass 5% One 100a 00 80a 20 80a 20
Two 80a 20 60b 40 60b 40
R.S.R. El-Mohamedy et al.
Bio-enhancing bagasse 10% One 60b 40 60b 40 40c 60

two 20d 80 0d 100 0e 100
Enhancing bagasse 5% One 100a 00 100a 00 100a 00
Two 100a 00 80a 20 80a 20
Enhancing bagasse 10% One 100a 00 100a 00 80a 20
Two 80a 20 80a 20 60b 40
T. harzianum 50 ml/L One 100a 00 100a 00 100a 00
Two 100a 00 60b 40 60b 40
T. harzianum 100 ml/l One 80a 20 80a 20 60b 40
Two 60b 40 60b 40 40 60
Plant guard 2 ml/L One 100a 00 100a 00 80a 20
Two 60b 40 60b 40 40c 60
Plant guard 4 ml/L One 80a 20 80a 20 60b 40
Two 40c 60 40c 60 20d 80
Control (check treatment) 100a – 100a – 100a –
Figures with the same letter in the same row are not significant at (P ¼ 0.05).
Results in Table 3 clearly show that the same trend of results was achieved. Two
soil applications caused a large effect in decreasing the severity of root rot disease
compared with one soil application as well as control treatment. Two soil
applications of artificially infested soil with M. phaseolina, F. oxysporium and F.
solani by bio-enhancing bagasse 10%, Plant Guand 4 ml/l and T. harzianum 100 ml/l
treatments caused a reduction of disease severity on grapevine seedlings reaching
100, 100 and 88.2%; 69.2, 57.1 and 52.9%; 46.2, 42.8 and 41.2% respectively. These
values of reduction were 69.2, 57.1 and 47.1%; 46.2, 35.7 and 29.4%; 46.1, 42.8 and
41.2% on grapevine seedlings inoculated with the same pathogens as with one soil
application.
The least effective treatments in decreasing severity of root rot disease incidence
were enhanced bagasse 5 and 10% (w/w); Plant Guard 2 ml/l and T. harzianum
50 ml/l, as there are no significant differences between these treatments and control
(non-treated seedlings), especially when these treatments were applied as a single soil
application.
Field experiments
Experiments were carried out under field conditions to evaluate the effect of
different biological soil treatments on recovering naturally infested grapevines with
root rot disease colonization of pathogenic fungi in roots, population density of
T. harzianum in rhizospher soil of treated vines, as well as fruit yield of vines was
estimated.
Results in Table 4 indicate that all tested soil treatments show a significant
decrease in percentages of disease severity after 30, 60 and 120 days with one or two
soil application treatments. Two soil applications with bio-enhancing bagasse at a
rate of 10% (w/w) of soil and plant Guard 4 ml/l treatments significantly reduced the
number of infested vines as well as the percentages of disease severity of these vines
after 30, 60 and 120 days from application.
Two soil applications with bio-enhancing bagasse 10% or plant caused 4 ml/l
treatments resulting in recovery of 3, 5, 6 and 1, 3, 4 vines and decreased disease
severity to 41.6, 30.5, 13.3% and 47.2, 41.6, 25.0% compared with 61.1, 69.4, 75.0%
for control (untreated vines) after 30, 60, 120 days from application respectively.
Meanwhile, a single application of the same treatments caused a recovery of 1, 2, 4
and 1, 2, 2, vines and decreasing disease severity of 41.6, 30.5% and 47.8, 38.8%
after 60 and 120 days from application.
On the other hand, bio-enhancing bagasse 5% and plant Guard at 2 ml/l had a
considerable beneficial effect in decreasing disease severity on infected trees
compared with control (untreated infected trees).
Effect of biological soil treatments on colonization of root rot pathogens in vine roots
and density of T. harzianum in rhizosphere soil
Feeder roots collected from treated and untreated trees were cut into small root
pieces (segments). After sterilization they were planted onto peptone PCNB medium
for isolation Fusarium spp. and PDA medium for isolation of M. phaseolina.
Results in Table 5 show that all tested treatments significantly reduced the
percentages of root segments yielding Fusarium or Macrophomina in one or two soil
applications after 60 and 120 days from application.
80
Table 3. Effect of different soil treatments on root rot disease severity on grapevine cv. Thompson seedlings transplanted into artificially infested soil in
greenhouse.
% root rot disease severity.

Soil treatment No. of soil application F. solani Reduction % F. oxysporium Reduction % M. phaseolinae Reduction %
Bio-enhancing bagasse 5% One 65b 23.5 55b 21.4 45c 30.8
Two 50d 41.2 40d 42.8 35d 46.1
Bio-enhancing bagasse 10% One 45d 47.1 30d 57.1 20e 69.2
Two 10f 88.2 00e 100 00f 100
Enhancing bagasse 5% One 85a 0.0 70a 0.0 60a 7.7
Two 80a 5.9 65a 7.1 55b 15.4
Enhancing bagasse 10% One 80a 5.9 65a 7.1 55b 15.4
Two 75b 11.8 60b 14.3 50c 23.1
T. harzianum 50 ml/L One 80a 5.9 65a 7.1 55b 15.4
Two 70b 17.6 55b 21.4 45c 30.8
T. harzianum 100 ml/L One 65c 23.5 50c 28.6 40c 38.5
Two 50d 41.2 40d 42.8 35d 46.2
Plant guard 2 ml/L One 75b 11.8 60b 14.3 50c 23.1
Two 60c 29.4 50c 28.5 40c 38.5
Plant guard 4 ml/L One 60c 29.4 45c 35.7 35d 46.2
Two 40e 52.9 30d 57.1 20e 69.2
Control (check treatment) 85a 0.0 70a 0.0 65a 0.0
Table 4. Development of root rot disease on grapevine cv. Thompson as affected by different soil treatments under field conditions in the Nobaria region.
Development of root rot disease

No. of diseased vineyards after application % disease severity after application
Soil treatment No. of soil application 0 day 30 day 60 day 120 day 0 day 30 day 60 day 120 day
Bio-enhancing bagasse 5% One 9a 9a 8b 7b 50.0a 52.7b 50.0b 44.4b
Two 9a 9a 8b 5c 50.0a 47.2bc 41.6c 35.0c
Bio-enhancing bagasse 10% One 9a 8b 7b 5c 50.0a 50.0b 41.6c 30.5d
Two 9a 6c 4d 3d 50.0a 41.6c 30.5d 13.8
Plant guard 2 ml/L One 9a 9a 8b 7b 50.0a 52.7b 52.7b 40.2b
Two 9a 9a 8b 6 50.0a 50.0b 44.4 30.5d
Plant guard 4 ml/L One 9a 8b 7b 7b 50.0a 52.7b 47.8bc 38.8b
Two 9a 8b 6c 5c 50.0a 47.2bc 41.6c 25.0e
Control 9a 9a 9a 9a 50.0a 61.1a 69.4a 75.0a
Archives of Phytopathology and Plant Protection
81
82
Table 5. Root colonization and Trichoderma propagule counts (cfu) in rhizosphere soil of grapevine cv. Thompson as affected by different soil
treatments under field conditions at Nobaria region.
Root colonization
Trichoderma propagules counts
% Pieces yield Fusarium spp after % Pieces yield M. phaseolina after (cfu) 6 105/g soil
Soil treatment No. of application 0 day 30 day 60 day 120 day 0 day 30 day 60 day 120 day 0 day 30 day 60 day 120 day
Bio-enhancing One 55a 48b 44b 40b 20a 17b 19b 18b 1.4b 2.5a 3.4b 3.8b
bagasse 5% Two 52a 41c 37c 22d 20a 15c 15c 10d 1.6a 2.8a 3.7b 4.0b
Bio-enhancing One 50a 43c 36c 26d 17b 16b 15c 11d 1.2c 2.6a 3.8b 4.6b
Bagasse 10% Two 48a 36d 27e 12f 21a 10d 12d 5e 1.5b 2.9a 4.1c 5.2c
Plant guard One 57a 46b 45b 38b 19b 17b 19b 18b 1.4b 2.5a 3.3 3.6
2 ml/L Two 51a 40c 40 26 21a 15c 16 12d 1.8a 2.6a 3.6b 4.0b
Plant guard One 55a 45b 42b 33c 22a 15c 18b 14c 1.5b 2.5a 3.6b 4.2b
4 ml/L Two 52a 41c 30d 18e 18b 14c 12d 11d 1.3c 2.7a 4.0c 4.8c
Control 54a 57a 62a 66a 24a 23a 26a 30a 1.8a 2.1a 2.6a 2.8a
The lowest percentages of root segments yielding Fusarium spp. were recorded
when bio-enhancing bagasse 10% and plant Guard 4 ml/l treatments were applied as
two soil applications. These values were 36, 27, 12% and 41, 30, 18% compared with
57, 62, 66% for control (untreated vines) after 30, 60, 120 days of application
respectively. Meanwhile, Macrophomina phaseolina was high affected by the same
treatments, however the percentages of infected roots were 10, 12, 5% and 14, 12,
11% compared with 23, 26, 30% for control after 30, 60, 120 days from application,
respectively.
In one soil application with the same treatments, the percentages of root
colorized by Fusarium spp. decreased to 43, 36, 26% and 45, 42, 33%, but those
colonized by Macrophomina spp. decreased to 16, 15, 11% and 15, 18, 14% after 30,
60, 120 days from application, respectively. On the other hand, bio-enhancing
bagasse 5% and Plant Guard 2 ml/l showed a moderate effect in reducing root
infection by Fusarium spp. and Macrophomina phaseolina.

Concerning the effect of different but biological soil treatments on population
density of Trichoderma spp., it is clear that all soil treatments cause an increase in the
population density of Trichoderma spp. Propagule count, which decrease in the
rhizosphere soil of untreated vines (control).
Bio-enhancing bagasse 10% and Plant Guard 4 ml/l treatments resulted in
increasing propagule counts of Trichoderma around roots of treated vines compared
with other treatments of one or two soil applications. These records were 2.9, 4.1,
5.2 6 104 cfu and 2.7, 4.0, 4.8 6 104 ctu/g soil with two soil applications and 2.6,
3.8, 4.6 6 104 ctu/g soil and 2.5, 3.6, 4.2 6 104 cfu/g soil with one soil application
compared with 2.1, 2.6, 2.8 6 104 ctu/g soil in untreated trees (control) after 30, 60,
120 days from application.
Effect of biological soil treatments on grapevine yield

Cluster and berry weight are important parameters for quality. Results in Table 6
revealed that all biological treatments cause a significant increase in yield of treated
vines compared with untreated vines (control). The highest values of clusters and
berries weight as well as yield/vine kg were obtained from vines treated twice with
Table 6. Effect of different soil treatments on no. of cluster per vine, cluster weight, berry
weight and yield/vine of grapevine cv. Thompson under field conditions in Nobaria region.
Yield Parameter of Thompson seedless grapevine

No. of soil No. of Cluster Berry Yield/vine Increase
Soil treatment application cluster weight (g) weight (g) (kg) %
Bio-enhancing One 13b 420a 2.5a 5.5b 27.9b
bagasse 5% Two 11a 525b 2.8a 5.8b 34.9c
Bio-enhancing One 12a 515b 2.9a 6.2c 44.2d
bagasse 10% Two 13b 500b 3.2b 6.5c 51.2e
Plant guard One 12a 460a 2.5a 5.5b 27.9b
2 ml/L Two 13b 450a 2.8a 5.8b 39.9c
Plant guard One 14c 440a 2.6a 6.2c 44.2d
4 ml/L Two 12d 530b 3.5b 6.4c 48.8e
Control 11a 390a 2.4a 4.3a 0.0a
bio-enhanced bagasse 10% and plant guard 4 ml/l. They recorded the value weights
of clusters, berries as well as yield per vine as much as 500, 3.2, 6.5 and 530, 3.5, 6.4,
respectively. Meanwhile these values were 390, 2.4 and 4.3 for untreated vines
(control). The large increase in yield vine was recorded on vines that were treated
with bio-enhancing bagasse 10% (51.2% increase over control) and Plant Guard
4 ml/l (48.8% increase over control) with two soil applications. Meanwhile, bio-
enhancing bagasse 5% and Plant Guard 2 ml/l treatments cause the lowest
percentage increase in vine yield with one soil application.
Discussion
Root rots are the most serious diseases affecting grapevine in nurseries and in fields.
Fusarium oxysporum, F. solani, Maccrophomina phaseolinae, Rhizoctonia solania and

Phytophthora spp. are considered as the main pathogens of theses diseases (Badawy
1973; Marais 1979; Mourad 1983; Walker 1992, 1994; Latorre et al. 1997; Mahrous
1994; Andrade et al. 1995; Ziedan 2003).
The present study revealed that T. harzianum and plant guard (4 ml/l) caused the
highest reduction 80.0, 83.3, 88.9% and complete reduction (100%) on linear growth
of F. oxysporium, F. solani and M. phaseolinae, respectively. In this regard T.
harzianum and biocides such as plant guard have been recorded in inhibition and
control of several soil borne plant pathogens (Ulkhede 1992; Ragab et al. 1999; El-
Mohamedy 2004). Using T. harzianum and/or plant guard for suppression and
control of soil borne plant pathogens has been reported by many investigators
(Ulkhede 1992; Ragab et al. 1999; El-Mohamedy 2004; Ziedan et al. 2005).
The antagonistic activities of T. harzianum are mediated by antibiotics, enzymes,
toxins, or direct parasitism. Also, the inhibitory effect of plant guard against
pathogens may be attributed to the effect of the original compound (T. harzianum
3 6 104 cfu/ml).
In greenhouse experiments, T. harzianum formulation on sugar cane bagasse
(bio-enhancing bagasse), spore suspension of T. harzianum and biocide (plant guard)
were applied as soil treatments for controlling root rot pathogens of grapevine. Two
soil applications of bio-enhancing bagasse 10% (w/w) of soil, plant guard 4 ml/l and
T. harzianum 100 ml/l treatments showed a large effect in decreasing the percentages
of root rot infection and severity on grapevines seedlings transplanted in soil
artificially infested with F. oxysporium, F. solani and M. phaseolinae. Meanwhile, the
same treatments gave a smaller effect when they were applied as a single soil
application with low dose (5%, 2 ml/l and 50 ml/l respectively). This may be
explained due to the low potential inoculum of the biocontrol agent (T. harzianum)
in soil (Guy and Baker 1977; Adams, 1990). Moreover, twice application of the same
materials at the high dose (10%, 4 ml/l and 100 ml/l) could lead to an increase in
potential inoculum of Trichoderma harzianum in the soil, and a decrease in the ability
of pathogens to infect and colonise seedling roots. This results support the findings
of Hadar et al. (1979), Elad et al. (1986), Loeffez et al. (1986), Nemec et al. (1996),
Prasad and Ragashwaran (1999), Liu and Huany (2000) and Tewari and
Mulkhapodhyay (2001). El-Mohamedy (2004) mentioned that Fusarium root rot
on mandarin seedlings was significantly reduced when T. harzianum alone or
formulated on sugar cane bagasse was delivered into artificially infested soil as two
applications.
Under field conditions, incorporation of T. harzianum formulated on sugar cane

bagasse at a rate of 10% and plant guard 4 ml/l into soil around the stem of
grapevines that are affected by root rot disease resulted in recovering a high number
of these diseased vines, decreasing the disease severity and causing a large reduction
in the percentages of root pieces colonized by either Fusarium spp. or M. phaseolinae.
Moreover, these treatments resulted in increasing the propagule counts (cfu) of
Trichoderma spp. in rhizospher soil of treated seedlings compared with untreated
(control). Meanwhile, at a low dose of plant guard (2 ml/l) and 5% of bio-enhancing
bagasse treatments, a moderate effect in controlling root rot disease was achieved.
The results obtained are in line with those of Yehia et al. (1994), Sawant et al. (1995),
Abdel-Kader (1997), Prasad and Ragashwaran (1999), Liu and Huany (2000),
Tewari and Mulkhapodhyay (2001), Sharma and Sarma (2002), Ziedan et al. (2005)
and El-Mohamedy et al. (2006).
Nemec et al. (1996) noted that amended planting mixes with formulations of
commercial biocontrol agents such as T. Harzianum, Bacillus subtilis, Gilocladium
virens and Streptomyces spp. reduced root rot and grown rot diseases on tomato, bell
pepper, celery and citrus. Control of soil borne plant pathogens through soil treated
with biocontrol agents and organic material such agricultural wastes may increase
the activity of indigenous microflora resulting in suppression of pathogen
populations by competition or specific inhibitors and/or inducing plant defense
mechanisms (Windham et al. 1986; Huang 1991; Mitra and Nandi 1994; Ceuster
et al. 1999; Liu and Huany 2000; Satish et al. 2000).
The reduction in root rot disease infection and severity on treated grapevines by
bio-enhancing bagasse and plant guard treatments may be explained by to the highly
decreased inability of pathogens to colonize vines roots especially after 60 and 120
days from application and increasing propagule counts of T. harzianum in
rhizosphere soil of treated vines compared with untreated control (Table 5).
Our results clearly show that applying bio-enhancing bagasse 10% and plant
guard 4 ml/l treatments to grapevine soil infected with root rot disease cause a large
increase in yield/vine total and weight of cluster as well as berry weight, if compared
with untreated vines (control). These increases may be explained due to controlling
propagules of root rot pathogens in rhizosphere soil and the decreasing rate of
disease severity and increasing population counts of T. harzianum in rhizosphere soil
of treated vines, restricting the development of feeder roots, providing a healthy and
vigorous vine capable of producing maximum yield. The same trend of results was
obtained by Elad et al. (1986),Ulkhede (1992), Yehia et al. (1994), Sawant and
Sarma (2002), El-Mohamedy (2004) and El-Mohamedy et al. (2006).
In conclusion, treating artificially or naturally infested soil with root rot
pathogens by Trichoderma harzianum formulated on agricultural wastes such sugar
cane bagasse or as biocide (plant guard) causes highly significantly reduced root rot
disease and increased yield and quality of grapevine yield. So, it can be suggested
that soil amended by such treatments may be safely used commercially as a
substitute for traditional fungicidal treatments.
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Biological soil treatment with Trichoderma harzianum to control root rot

Article in Archives of Phytopathology and Plant Protection · January 2010

Riad el-mohamedy El Sayed Hussein Ziedan

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Biological soil treatment with Trichoderma harzianum to control root rot

Plant Pathology Department, National Research Centre, Giza, Egypt

Augmentation of soil with diﬀerent biological treatments i.e., Trichoderma

*Corresponding author. Email: riadelmohamedy@yahoo.com

ISSN 0323-5408 print/ISSN 1477-2906 online

soil treatments as a substitute for chemical fungicides is considered very important,

Materials and methods

Biocontrol agent and biocides

Eﬀect of T. harzianum and plant guard on root rot pathogens in vitro

Bio-enhancing and enhancing bagasse

– Bio enhancing bagasse: 50 and 100 g/kg soil.

(1) Bio-enhancing bagasse at the rate of 0.5 and 1.0 kg/vine

(2) Plant Guard at the rate of 2 and 4 ml/l

Eﬀect of soil treatments on the colonization of pathogens in grapevine roots and

Eﬀect of biological soil treatments on grapevine yield

and 88.9%, respectively.

Table 1. Linear growth reduction of grapevine root rot pathogens as aﬀected by T.

*L: Linear growth (mm). **R: % Reduction.

% root rot disease infection.

Bio-enhancing bagasse 10% One 60b 40 60b 40 40c 60

% root rot disease severity.

Development of root rot disease

infection by Fusarium spp. and Macrophomina phaseolina.

Eﬀect of biological soil treatments on grapevine yield

Yield Parameter of Thompson seedless grapevine

Fusarium oxysporum, F. solani, Maccrophomina phaseolinae, Rhizoctonia solania and

Under ﬁeld conditions, incorporation of T. harzianum formulated on sugar cane

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