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Citrus compost and its water extract for cultivation of melon plants in
greenhouse nurseries. Evaluation of nutriactive and biocontrol effects
A. Bernal-Vicente
a
, M. Ros
a
, F. Tittarelli
b
, F. Intrigliolo
c
, J.A. Pascual
a,*
a
Department of Soil Water Conservation and Organic Waste Management, Centro de Edafologia y Biologia Aplicada del Segura (CEBAS-CSIC), P.O. Box 164, 30100 Espinardo,
Murcia, Spain
b
Centro per la Ricerca e la sperimentazione in Agricoltura, Centro di ricerca per lo studio delle relazioni tra pianta e suolo (CRA-RPS), via della Navicella, 2, 00184 Rome, Italy
c
Centro per la Ricerca e la sperimentazione in Agricoltura, Centro di ricerca per l’Agrumicoltura e le Colture Mediterranee (CRA-ACM), corso Savoia 190, 95024 Acireale (CT), Italy
article info
Article history:
Received 2 November 2007
Received in revised form 7 April 2008
Accepted 8 April 2008
Available online 21 May 2008
Keywords:
Fusarium oxysporum
Compost
Biocontrol
Melon plant
abstract
Two different types of citrus composts, and their water extracts, were tested with regard to their utilisa-
tions as partial substitutes for peat in growing media for melon seedlings in greenhouse nurseries. Both
compost showed higher plant growth than peat. Compost composed by citrus waste and green residue
(C2) showed greater plant growth than compost obtained from the same organic matrices mentioned
above further the addition of sludge obtained from citrus industry (C1). Compost C2 showed a greater
auxinic effect than C1 and it was the only one that showed cytokinic effect. Both composts also demon-
strated a biocontrol effect against Fusarium oxysporum for melon plants: the effects were also higher in C2
than in C1. Higher number of isolated fungi was active against F. oxysporum in compost C2, than compost
C1. No different bacterial biocontrol efficacy was observed between both composts. The water extracts of
both composts gave lower plant yields than their solid matrices, their relative effects being similar to
those of the solid composts (C2 extract gave higher plant yields than the extract from C1). The biocontrol
effects of compost water extracts followed the same trend.
Ó2008 Elsevier Ltd. All rights reserved.
1. Introduction
Fusarium oxysporum is a fungus which causes many different
diseases of cultivated plants. One of the most important is Fusarium
wilt of melon plants (Armstrong and Armstrong, 1981; Arnon,
1949). Chemical methods used to control vascular wilt are either
not very efficient or imply negative effects on environmental and
human health (Brimner and Boland, 2003; Becker and Schwinn,
1993; Boutler et al., 2000; Hoitink and Boehm, 1999). Biological
control of pests has developed during recent years as a response
to the increasing demand for alternatives to the chemical control
of plant pathogens (Abawi and Widmer, 2000; Garcia et al., 2004;
Pascual et al., 2002).
The use of peat as a major constituent of potting media in
greenhouse nurseries is challenged by economic and environmen-
tal pressures (Zhang et al., 2004). The use of specific compost as
partial alternative to peat would permit lower costs by themselves
and lower dependence of chemical inputs, due nutrient content,
and an important biopesticide effect, reducing also costs of green-
house plant manipulation (Hoitink and Fahy, 1986; Pascual et al.,
2002; Ros et al., 2005). The mechanisms involved in the suppres-
sion of pathogens are variable depending of each type of composts
and their physical, physico-chemical, chemical and biological char-
acteristics and also on the type of plant pathogen to be controlled
(El-Masry et al., 2002; Boutler et al., 2000; Hoitink and Boehm,
1999). Therefore, the choice of the method for controlling plant
pathogens should be done on a case-by-case basis, taking into ac-
count the pathogen physiology and its potential vulnerability in
one or more of its phases of development (Pascual et al., 2002).
Composting of organic residues from the citrus processing
industry has been investigated during recent years in relation to
the environmental issues connected with the increasing amount
of citrus fruits processed yearly (Calabretta et al., 2004; Tittarelli
et al., 2002). Soil-less culture, the economic importance of which
is increasing year by year all over the world, represents a challenge
for the utilisation of alternative composts as partial or integral sub-
stitutes for a non-renewable resource such as peat (Tittarelli et al.,
2003). Citrus composts showed a strong correspondence to the
characteristics of previously-studied suppressive organic materi-
als, regarding their physico-chemical and chemical characteristics
(high level of lignocellulosic compounds, high C/N ratio and pH
(Chef et al., 1983; Serra-Wittling et al., 1996; Hoitink and Boehm,
1999; Cotxarrera et al., 2002; Rose et al., 2003); encouraging dee-
per research into the potential biopesticide use of citrus compost
as components of soil-less growing media.
The main objective of this research was focused on demonstrat-
ing the potential use of citrus composts as partial peat substitutes
0960-8524/$ - see front matter Ó2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2008.04.019
*Corresponding author. Tel.: +34 968396339; fax: +34 968396213.
E-mail address: jpascual@cebas.csic.es (J.A. Pascual).
Bioresource Technology 99 (2008) 8722–8728
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
for producing melon (Cucumis melo L.) plants, under natural and in-
duced F. oxysporum f. sp. melonis pressure, in greenhouse nurseries.
In particular, compost produced from citrus wastes, pruning mate-
rials and agro-industrial sludge was compared with citrus compost
produced without sludge (the absence of sludge in the starting mix-
ture is required by the most-widely used protocol of compost pro-
duction in organic farming), in terms of their potential biological
control of F. oxysporum. Furthermore, water extracts from both
composts were also investigated in order to evaluate the feasibility
of their use in peat fertigation.
2. Methods
2.1. Compost production
Citrus composts were produced in the Azienda Sperimentale
Palazzelli (Lentini), of the CRA-ACM (CT, Italy). Two piles of 9 m long,
1.5 m wide and 0.8 m high was prepared, treating each 3 m of heap
length (sub-pile) as a replicate. The first pile was composed by 40% of
citrus wastes (a mixture of citrus pulp and skins from the citrus
industry), 20% sludge obtained from citrus industry waste-water
treatment and 40% green residues (pruning material from citrus
trees and ornamental shrubs) (C1). The second pile was composed
only by 60% citrus wastes and 40% green residues, without sludge
(C2). The specific composting process was described by Tittarelli
et al. (2003). At the end of the process, 100 kg of compost from each
sub-pile, were collected randomly, homogenised and sieved at
10 mm. Fifteen kilograms from each sub-pile were stored at 4 °C
until its utilisation for the assays. The main physico-chemical and
chemical parameters of the composts are reported in Table 1.
2.2. F. oxysporum isolation and infection procedure
F. oxysporum was isolated from infected melon plants obtained
from a greenhouse nursery (Nash and Snyder, 1962) and stored in
potato dextrose agar (PDA). For producing F. oxysporum for the as-
say, a potato dextrose broth (PDB) was inoculated with the isolated
F. oxysporum and incubated at 25 °C for 5 days.
2.3. Experimental design
2.3.1. In vitro assay
2.3.1.1. Compost bacteria and fungi isolation. Both composts were
suspended on sterilised Ringer solution (1/10 w/v) and 1/10 serial
dilutions were done to calculate the number of bacterial colony
forming units (CFU) by plating them on 1/10 tryptose soya agar
(TSA, oxoid 3 g L
1
, agar 15 g L
1
, nystatin 50 mg L
1
); and to cal-
culate the fungal CFU by plating them on PDA (Oxoid, 39 g L
1
, rose
bengal 50 mg L
1
, streptomycin 100 mg L
1
). Furthermore, each
different type of bacterial and fungal colony, according to visual
parameters (colour, shape, type of growth, etc.), was isolated and
preserved on slant TSA and PDA, respectively.
2.3.1.2. Bacterial antagonism against F. oxysporum. Each isolated
bacteria was placed in the middle of a PDA plate, drawing a line
from one to the opposite side. After 1 day of incubation at 25 °C,
a PDA disc (5 mm) of F. oxysporum was placed at 2 cm length from
the bacterial solution line, incubated at 25 °C for 7 days. A control
with F. oxysporum was placed in a Petri dish without any bacteria.
Positive biocontrol bacteria were considered when the F. oxyspo-
rum colony size was smaller than 50% of the control. Antibiotic
antagonism effect was considered when pathogen mycelium did
not grow till the bacteria line. Mycoparasitism antagonism effect
was considered when interaction between F. oxysporum and the
tested bacteria was observed.
2.3.1.3. Fungal antagonism against F. oxysporum. Two PDA discs
(5 mm), one with F. oxysporum and the other with the tested iso-
lated fungi, were placed at 4 cm distance in a PDA plate, incubated
at 28 °C for 7 days. A control with F. oxysporum was placed in a Pet-
ri dish without any fungi. A positive biocontrol fungus was consid-
ered when F. oxysporum colony size was smaller than 50% of the
control. Antibiotic antagonism effect was considered when patho-
gen mycelium did not grow till the tested fungi. Mycoparasitism
antagonism effect was considered when interaction between F.
oxysporum and the tested fungi was observed.
2.3.1.4. Abiotic effect of compost against F. oxysporum. In order to
evaluate the abiotic component of compost action against F. oxy-
sporum growth and sporulation, PDA medium was prepared by
autoclaving a double concentration of PDA (78 g L
1
), reaching
the normal PDA concentration (39 g L
1
) by adding compost water
extracts, once it was autoclaved and cooled. Sterilised compost ex-
tracts were obtained by mixing a 1:5 (w:v) ratio (compost:distilled
water) for 12 h at room temperature. PDA with distilled water was
also prepared to be used as control.
For testing the compost extract effect on F. oxysporum growth, a
PDA disc (5 mm) of F. oxysporum was placed in the middle of a Petri
dish and incubated at 28 °C, measuring the colony size after 7 days.
For testing the effect on F. oxysporum spore germination, the above
protocol was used to prepare the different media, adding rose ben-
gal (50 mg L
1
), plating a known F. oxysporum spore solution, and F.
oxysporum CFU was counted after 7 days.
2.3.1.5. Overall (abiotic and biotic) effect of compost against F.
oxysporum. F. oxysporum growth and spore germination was also
tested in non-sterile conditions, following the above-mentioned
protocol. The results obtained in this experiment minus the abiotic
effect obtained above could be attributed to the biotic effect of the
composts, assuming that synergic effect between abiotic and biotic
components was negligible.
Another assay was prepared, cutting, in sterile conditions, a
2-cm-wide strip of PDA in Petri dishes. The gap was filled with a
mixture of vermiculite plus compost (1:1, v/v), moistened with
1 mL of sterile distilled water. A control was also made by mixing
vermiculite with potato dextrose broth (1:1, v/v), filling the gap. A
disc containing the pathogen was put on one side of the filled gap
and incubated at 28 °C for 7 days. The capacity of F. oxysporum to
cross the artificial strip created in the Petri dishes was evaluated
by measuring the size of the mycelium developed, according to
the following qualitative criteria: 0, the pathogen did not reach
the organic barrier; 1, it reached the organic barrier; 2, it reached
Table 1
Physico-chemical and chemical characteristics of the organic materials used and their
standard deviations (values are referred to dry matter at 105 °C)
Parameter Peat C1 C2
pH 6.34 ± 0.25 8.4 ± 0.05 8.5 ± 0.07
Total organic carbon (%) 49.4 ± 1.4 31 ± 0.8 38 ± 1
Total N (%) 0.97 ± 0.07 2.8 ± 0.1 2.5 ± 0.1
P
2
O
5
(%) 0.7 ± 0.1 2.3 ± 0.1 0.7 ± 0.1
K
2
O (%) 0.6 ± 0.1 0.8 ± 0.1 0.7 ± 0.1
C/N 50 ± 3 12 ± 0.5 15 ± 1
Humic and fulvic acid C (%) 25 ± 1.5 14 ± 1 18 ± 1
Total cadmium (mg/kg) <0.1 1.5 ± 0.1 <0.1
Total mercury (mg/kg) <0.1 <0.1 <0.1
Total copper (mg/kg) 12 ± 1 37 ± 4 32 ± 4
Total zinc (mg/kg) 90 ± 3 320 ± 12 99 ± 6
Total nickel (mg/kg) 12 ± 2 31 ± 2 20 ± 2
Total lead (mg/kg) 5 ± 1 10 ± 1 13 ± 1
Hexavalent chromium (VI) (mg/kg) n.d.
a
n.d.
a
n.d.
a
Electrical conductivity (mS/cm) 0.51 ± 0.04 2.08 ± 0.08 1.78 ± 0.09
a
n.d. = not detectable.
A. Bernal-Vicente et al. / Bioresource Technology 99 (2008) 8722–8728 8723
the organic barrier but did not cross it completely; 3, it crossed
completely the organic barrier (Table 2).
2.3.1.6. Analysis of soil microbial community structure by denaturing
gradient gel electrophoresis (DGGE). For DGGE analysis, total DNA
was extracted from compost using a FAST DNA Spin kit (BIO 101,
USA). After extraction, bacterial and fungal DNA was amplified in
a PCR thermocycler (PCR Express, ThermoHybaid), by using specific
primer sets for 16S rDNA (F338/R907) and 18S rDNA (FR1/FF390),
respectively (Vainio and Hantula, 2000). PCR products for fungi and
bacteria were run on a denaturing gradient gel electrophoresis
(DGGE) using the DCODE TM Universal Mutation Detection system
(BioRad laboratories, Inc.). The DGGE fingerprint pattern was
scanned and analysed with GelCompar software package (version
3.50; Applied Maths, Ghent, Belgium). Band sharing was quantified
as a Dice similarity coefficient, followed by Cluster analysis con-
ducted by the Ward method (Ward, 1963). The experiment was
carried out in triplicate to allow statistical comparisons between
treatments.
2.3.1.7. Bioassay to detect the presence of auxin and cytokinin-like
compounds. For the auxin bioassay, oat seeds were germinated for
72 h in dark conditions and the apices of the coleoptiles were cut at
5 mm lengths and immersed in a buffered solution (0.01 M
KH
2
PO
4
, pH 4.5) of 2% (w/v) sucrose for 2 h, in order to eliminate
endogenous auxin. Ten cut coleoptiles were placed in a Petri dish,
in triplicate, and incubated with 5 mL of different compost extract
dilutions, in dark conditions, for 24 h, considering dilution 0, the
initial compost water extract (1/5; w/v). Section length was mea-
sured at each dilution and a growth curve was drawn, comparing
it with a typical auxin curve obtained using 0.01 M indolacetic
acid. It is known that auxinic compounds, at specific concentra-
tions, stimulate coleoptile growth, while, at higher concentrations,
show an inhibitory effect (Barcelo et al., 1995).
For the cytokinin bioassay, 0.1 g of equally-sized pieces of let-
tuce were incubated for 7 days, in the dark at 25 °C, in different
dilutions of the compost water extracts, considering dilution 0,
the initial compost water extract (1:5; w/v). After incubation, chlo-
rophyll was extracted from each leaf sample and determined by
the method of Arnon (Bruinsma, 1963). Cytokinin-like compounds
inhibit the degradation of chlorophyll at specific concentrations,
but stimulate chlorophyll degradation at higher concentrations
(Barcelo et al., 1995). Compost water extracts were compared with
a curve of diluted 0.01 M kinetin.
2.3.2. In vivo assay
The nutriactive and biocontrol capacity of compost and their
water extracts were assayed in vivo on melon seedlings under
the same operative commercial greenhouse nursery conditions.
Growing media were prepared by mixing each compost, C1 and
C2, with peat. Water extracts from compost C1 (EC1) and compost
C2 (EC2) were obtained by mixing compost and distilled water in a
1/5 (w/v) ratio, shaking the mixture for 12 h at room temperature
and filtering the supernatant through a mesh before use. In order
to study nutriactive and biocontrol capacity independently, it
was necessary to develop the experiment with and without patho-
gen pressure.
2.3.2.1. Nutriactive evaluation. Specific nursery plant polystyrene
containers, with 10 individual wells, were filled with different
prepared growing media. Each specific container was considered
as a unit and three replicates of each treatment were used. Cit-
rus compost treatments were performed with citrus composts–
peat mixtures (1/4 w/w). Compost water extract treatments
were studied on peat based growing media which differed from
the control (peat based growing media) as the solutions were
added during the experiment as described below. Two hundred
mL of distilled water were added to each compost–peat treat-
ment, in three times a week. Compost water extract treatments
received 200 mL of the specific compost water extract and con-
trol peat received the same amount of a nutrient solution, to
overcome the nutritional gap between compost–peat and peat
based growing media prepared in accordance with fertilisation
pattern for commercial melon seedling production. Ten melon
seeds were sown in each container and put in a germination
chamber until seedling emergence. After 60 h, each individual
container with pre-germinated seeds was moved, in a random
design, to a growth chamber with a 16-h photoperiod and a
day/night temperature regime of 21 °C/15 °C, with relative
humidity maintained at 70%.
2.3.2.2. Biocontrol evaluation. The same experimental design was
followed to evaluate the potential biocontrol capacity of the com-
posts and their extract, with the exception that the growing media
were infected by F. oxysporum, using an inoculation size of
2.9 10
4
UFC per gram.
Plants were harvested three times: after 30, 37 and 43 days,
measuring plant fresh weight. The total incidence of the different
organic treatments was calculated as the increased percentage of
melon plants weight with respect to the mean obtained with peat
(control) in the presence of the pathogen. The overall incidence of
the treatments compared was the result of the combination of
nutriactive effect, as consequence of the nutrient and biostimulat-
ing compound incorporation, and biocontrol effect due to abiotic
and biotic components. The nutriactive effect was calculated as
the increased percentage of weight in melon plants grown with
the different treatments with respect to control peat in the absence
of the pathogen. On the other side, the biocontrol effect was calcu-
lated by subtracting from 100 (percentage value considered as ref-
erence for melon plant weight in absence of the pathogen for each
individual treatment), the pathogen incidence (percentage of mel-
on plant weight loss in presence of the pathogen respect to the
melon plant weight in absence of the pathogen, for each individual
treatment).
2.4. Statistical analysis
All the experiments were carried out by triplicate to establish
statistical comparisons among treatments. Mean values recorded
were subjected to one-way analysis of variance (ANOVA). To com-
pare the differences between specific treatments, the Tukey test
was used. The SPSS 11.0 software package was used for statistical
tests.
Table 2
Mycelium growth, spore germination of F. oxysporum with compost water extracts
(sterilised and non-sterilised)
Treatments Peat C1 C2
Mycelium growth (diameter, cm) Sterile 5.46b 5.27b 5.37b
Non-sterile 5.55b 4.18a 4.00a
Spore germination (CFU mL
1
) Sterile 3.7 10
6
a 3.4 10
6
a 3.5 10
6
a
Non-sterile 3.8 10
6
a 2.0 10
6
a 2.4 10
6
a
Organic barrier
a
Sterile 3 3 3
Non-sterile 2 1 0
Capacity of compost to behave as organic barrier to inhibit F. oxysporum.
a
The capacity of F. oxysporum to cross the artificial strip was evaluated according
to the following qualitative criteria: 0, the pathogen mycelium did not reach the
organic barrier; 1, reached the organic barrier; 2, reached the organic barrier but did
not cross it completely; 3, crossed completely the organic barrier. Values with the
same letters are not significantly different (P< 0.05).
8724 A. Bernal-Vicente et al. / Bioresource Technology 99 (2008) 8722–8728
3. Results and discussion
The main physico-chemical and chemical parameters of the fi-
nal products are reported in Table 1. Peat parameters are within
the range of values normally found for peat utilised as an organic
component of plant nursery substrates. Both composts showed
lower total organic carbon and higher pH and electrical conduc-
tivity than peat. C1 showed a lower total organic carbon content
and higher nutrient content than C2, due to the presence of
sludge in the starting pile mixture. The sludge incorporated a
higher amount of easily-mineralisable carbon and mineral com-
ponents, producing a high mineralisation rate during composting,
that reduced organic matter and concentrated nutrient content
(Tittarelli et al., 2002).
3.1. In vitro experiments
In vitro experiments were carried out in order to evaluate the
suppressive effect of both citrus composts and their water extracts
on F. oxysporum. Moreover, the experimental design was defined in
order to separate the roles of the abiotic and biotic components of
the composts in pathogen control.
Non-significant differences among the sterilised water extracts
of citrus composts and the control (distilled water) were reported
for mycelium growth and spore germination of F. oxysporum (Table
2). In non-sterile conditions, both citrus composts (C1 and C2) re-
duced significantly, with respect to the control, F. oxysporum myce-
lium growth, but not spore germination (Table 2). Compost C2, as
physical organic barrier, showed more evident inhibitory effects on
the pathogen than C1 (Table 2).
The number of bacteria and fungi, expressed as CFU, were an or-
der of magnitude higher in C1 than in C2. Number of different
types of bacteria colonies (attending colour, morphology, shape,
etc.) was similar for both citrus composts but the number of differ-
ent fungi colonies was higher for C2 than for C1 (Table 3).
From the biocontrol test against F. oxysporum, more than 60% of
isolated fungi in compost C2 were active against the pathogen,
while only 20% in compost C1 (Table 3). Fungi isolated from C2
showed a higher percentage of mycoparasitism than those of C1,
while the antibiosis effect was almost similar (Table 3). On the
other hand, the percentage of isolated bacteria with an active bio-
control effect against F. oxysporum was almost similar in both com-
posts, showing only antagonism, as a consequence of the
production of antibiotic-like compounds (Table 3).
The PCR-DGGE, obtained after cluster analysis to infer similari-
ties among the different banding patterns approach by using the
Pearson correlation coefficient and clustering by Ward, confirmed
the Petri dish results, showing lower similarity for bacteria (75%)
than for fungi (95%) among both citrus composts, marking the
important role for bacteria of sludge as primary organic matrix,
used in compost C1. Furthermore, genetic similarity among peat
and the two citrus composts was higher for fungi (about 70%) than
for bacteria (about 20%), that could be explained by lignocellulose
compounds, common organic matrices in both composts and peat,
are more important for fungi than for bacteria, than other inputs
such sludge (Mitali et al., 2007).
In sterile conditions, the in vitro assays showed no differences
between both composts and peat (Table 2). Such results were
shown also by other authors who demonstrated the lack of an
antagonistic effect of sterile water extracts against the tested path-
ogen (El-Masry et al., 2002). However, these results are not in
accordance with those of other authors, who reported some abiotic
effects of compost on F. oxysporum as a consequence of high levels
of nitrogen or physico-chemical changes following compost
amendment (Alabouvette, 1999; Jones and Woltz, 1981). It is pos-
sible that the abiotic factors of the assayed composts were not
powerful enough to have a direct effect on pathogen development
(Serra-Wittling et al., 1996).
On the other hand, in non-sterile conditions, both C1 and C2
water extracts and the organic barrier in Petri dishes appreciably
reduced F. oxysporum with respect to the control (Table 2). This
means that the studied composts should provide antagonistic
microorganisms (the main difference between non-sterile and
sterile conditions) against F. oxysporum (McQuilken et al., 1994;
El-Masry et al., 2002; Pascual et al., 2002; Zhang et al., 1998).
It can be hypothesised that the protective effect of the studied
composts was a combination of extracellular lytic enzyme pro-
duction (mycoparasitism) and antibiotic production, as reported
in Table 3 (Baziramakenga and Simard, 1998; Cronin et al.,
1996; Yohalem et al., 1996). The use of sludge as a matrix for
C1 compost production resulted, however, in a lesser biocontrol
effect than for C2, as a consequence of the reduction of suppres-
sive microorganisms, mainly fungi (Table 2). The addition of
sludge to the composting mixture (citrus plus green residues)
would incorporate or permit the growth of rapid-growth-strategy
microorganisms that inhibit the growth of lignocellulolytic
microorganisms, characterised by a slow growth strategy and
considered responsible for the biocontrol effect (Pascual et al.,
1997, 2002; Ros et al., 2006).
The in vitro assay also showed that the biopesticide effect
against F. oxysporum was attributed mainly to fungi (Table 3). Fun-
gi have the capacity to distribute their mycelia, occupying a greater
substrate surface and more ecological niches than bacteria, and
thus have more chance of interacting with plant pathogens (Mitali
et al., 2007). Furthermore, fungi showed two different biocontrol
mechanisms, mycoparasitism and production of antibiotic-like
compounds, while bacteria only showed the latter (Table 3)(Shen,
2000). As C2 showed greater antagonism than C1, demonstrated by
reduced F. oxysporum mycelium growth and by its low capacity to
cross the artificial organic barrier (Table 2), it can be assumed that
the main reason for F. oxysporum control was the presence of spe-
cific antagonistic fungi.
3.2. In vivo experiment
The effect of the growing media on plant growth under patho-
gen pressure demonstrated that citrus composts and their water
extracts (except EC1) produced significantly higher melon plant
weight than the control peat at all plant sampling times (Table
4). Plant weight was significantly higher on compost C2 than on
C1, and both composts showed significantly higher plant weights
than their water extracts (Table 4). In general, the total weight
Table 3
Fungal and bacterial CFU, number of different type of colonies, percentage of those
with specific activity against F. oxysporum and percentage for each type of antagonist
mechanism (antibiosis and mycoparasitism)
C1 C2
Bacteria (CFU g
1
) 5.7 10
8
b 3.2 10
7
a
Fungi (CFU g
1
) 8.3 10
5
b 4.7 10
4
a
Number of different type of colonies*
1
Different bacteria 10 13
Different fungi 12 8
Specific activity against F. oxysporum of each of the isolated
Bacteria (% respect total isolated) 10%a 15%a
Fungi (% respect total isolated) 20%a 62%b
Biocontrol mechanisms of each positive antagonist fungi
Antibiosis 95%a 83%a
Mycoparasitism 45%a 66%b
Values with the same letters are not significantly different (P< 0.05).
A. Bernal-Vicente et al. / Bioresource Technology 99 (2008) 8722–8728 8725
increase was 250% for compost C2 and around 150% for compost
C1 and EC2, at the end of the experiment.
The results obtained can be attributed to two differentiated and
specific effects: nutriactive (nutritional and bio-stimulation as-
pects) and biocontrol (reduction of the pathogen incidence).
3.3. Nutriactive effect
In absence of pathogen pressure, nutriactive effect at 43 days
was 78% higher than for peat for both composts, and about 25%
higher for EC2 (Table 5). Differences in plant growth between
compost treatments and peat could be attributed to the in-
creased plant nutrient content of the substrate in comparison
with peat. However, differences between the two composts and
their water extracts, (plant growth on C2 was greater than on
C1) cannot be explained by a nutrient effect because the nutrient
content was similar for the two composts or was even higher for
C1, the opposite of the trend observed for plant growth (Table
5).
The presence of auxinic and cytokininic-like compounds was
tested by specific bioassays to demonstrate the potential nutriac-
tive effect of both citrus composts. C2 showed both (auxinic and
cytokininic) effects, while C1 only showed auxinic effect (Figs. 1
and 2). Furthermore, C2 showed a greater auxinic effect than C1.
These hormone-like compounds would explain differences in plant
growth between the two composts (Table 5), underlining their
important role in initial plant growth stages (Atiyeh et al., 2002),
where differences in the nutriactive effect were more evident,
decreasing with time (Table 5). Water extracts from the composts
showed a significantly lower nutriactive effect than the solid com-
posts (Table 5), mainly because hormone-like compounds would
degrade easily with time and also they would not be totally
water-extractable due to their high molecular weight and the com-
plexes formed with humic substances (Atiyeh et al., 2002). EC2
showed a higher nutriactive effect than EC1, due mainly to the
greater amount of nutriactive compounds in C2 compared with
C1 (Figs. 1 and 2).
3.4. Biocontrol effect
The in vivo assay confirmed the in vitro results, citrus composts
showing a biocontrol effect, once the nutriactive effect was sub-
tracted, by reducing the effect of the pathogen on melon plants
(Table 6). This effect can be attributed to the biotic component of
the composts, as demonstrated by the in vitro assay (Table 2).
The biocontrol effect could be attributed also to niche competition
or induction of plant resistance (not tested in the in vitro assay)
(Alabouvette et al., 2006). Compost C2 and its water extract again
produced less pathogen incidence than C1, related mainly to a
higher content of specific microbiota for this purpose, as the
in vitro assay demonstrated.
The balance of the two implicated effects, biocontrol and nutri-
active, was calculated by dividing the biocontrol effect (by sub-
Table 4
Melon plant fresh weight at different sampling times (30, 37 and 43 days), in
F. oxysporum infected growing media
Treatments 30 days 37 days 43 days
Fresh weight, g (percentage of increase with respect to the
control peat)
Peat 1.22a (–) 1.28a (–) 1.30a (–)
C1 2.71d (122%) 2.30b (79%) 3.36b (158%)
C2 4.3e (252%) 4.26c (133%) 4.53c (251%)
C1 extract (EC1) 1.65b (35%) 1.26a (2%) 1.29a (1%)
C2 extract (EC2) 2.23c (83%) 2.28b (78%) 3.33b (156%)
Total pathogen incidence, in brackets.
At each sampling time, values with the same letters are not significantly different
(P< 0.05).
Table 5
Melon plant fresh weight at different sampling times (30, 37 and 43 days), in non-
infected growing media
Treatments 30 days 37 days 43 days
Fresh weight, g (percentage increase with respect to the control
peat)
Peat 1.93a (–) 2.53ab (–) 3.00a (–)
C1 3.20c (66%) 3.76c (48%) 5.36c (78%)
C2 4.51d (133%) 4.46d (76%) 5.35c (78%)
C1 extract (EC1) 2.21a (14%) 2.23a (12%) 2.74a (9%)
C2 extract (EC2) 2.54b (32%) 2.99b (18%) 3.76b (25%)
Nutriactive effect, in brackets, is the weight increase percentage respect to control
peat.
At each sampling time, values with the same letters are not significantly different
(P< 0.05).
0.5
0.625
0.75
0.875
1
0 -1-2-3-4-5-6-7-8-9-10
dilution
cm
C1 C2 IAI Control
Fig. 1. Auxinic-like effect of compost C1 and C2 compared with indolacetic acid
(IAI).
0.4
0.8
1.2
1.6
0 -1-2-3-4-5-6-7-8-9
dilution
mg clorophyll g-1
C1 C2 kinetin control
Fig. 2. Cytokininic-like effect of compost C1 and C2 compared with kinetin.
8726 A. Bernal-Vicente et al. / Bioresource Technology 99 (2008) 8722–8728
tracting from 100 the pathogen incidence percentage) by the nutri-
active effect. C2 showed a more balanced (nearly 1) nutriactive-
biocontrol effect than C1 (C2: 1.09; C1: 0.80). Compost water ex-
tracts demonstrated a lower overall effect than the solid organic
materials, mainly due to the low nutriactive effect of water-ex-
tracted compounds, with an unbalanced biocontrol-nutriactive ef-
fect (EC1: 5.39; EC2: 3.56).
4. Conclusions
The most important remarks are that composts produced from
organic matrices, such as citrus residues and pruning material
from citrus trees and ornamental shrubs, are a real alternative
to peat. Their use would permit reduced peat and agrochemical
costs due to their nutriactive effects (contents of nutrients and
hormone-like compounds) and their biocontrol effect (mainly
due to the biotic component). Compost made with the mixture
of citrus residues and green residues is adequate for plant seed-
ling production, according to the most-widely accepted protocol
of organic farming, while these organic matrices mixed with
sludge, for composting, can be utilised only in a protocol for con-
ventional nursery production. Compost water extracts can be
used for peat fertigation, even though both the nutriactive and
biocontrol effects were significantly lower with respect to the so-
lid composts.
Acknowledgements
The authors wish to acknowledge Dr. David J. Walker (IMIDA,
Murcia, Spain) for his correction of the written English.
The co-author Dr. Fabio Tittarelli wishes to acknowledge the
support of the OECD’s Cooperative Research Programme: Biological
Resource Management for Sustainable Agriculture Systems in the
funding of his research.
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Table 6
Pathogen incidence at different sampling times (30, 37 and 43 days), calculated as
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Percentage (%)
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C1 15.24b 38.91bc 37.29b
C2 4.44a 4.47a 14.77a
C1 extract (EC1) 24.93c 43.69c 52.89c
C2 extract (EC2) 12.16ab 23.52b 11.11a
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