Journal of Phytopathology and Pest Management 9(1): 1-13, 2022
pISSN: 2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Najwa Benfradj,
E-mail: najwabenfradj@yahoo.fr
1
Copyright © 2022
Comparison between two methods of mycelia growth
evaluation of some Oomycetes species
Najwa Benfradj*, Amani Rouini, Naima Boughalleb-M’Hamdi
Department of Biological Sciences and Plant Protection, High Institute of Agronomy of Chott
Mariem, University of Sousse, 4042 Sousse, Tunisia
Abstract
Keywords: calculated area, measured area, growth factors, mefenoxam, ratio.
Oomycetes pathogens causes devastating crop diseases worldwide. In this study,
we compared mycelium growth of 7 Oomycetes species using the calculated area
(CA) method obtained by perpendicular diameter of colony and measured area
(MA) method. Results revealed a significant difference between Oomycetes species
inhibition ratio using CA and MA, at lower mefenoxam concentrations. The
interval of variation between MA/CA methods was variable according to
Oomycetes specie. The largest interval (0.01-1000 μg/ml) was detected with
Pythium aphanidermatum and Phytopythium mercuriale. The second interval
(0.01-100 μg/ml) was determined by Phytophthora cryptogea, P. aphanidermatum,
P. ultimum and P. mercuriale. An interval ranged from 0.01μg/ml to 31.6μg/ml was
recorded with P. cryptogea and Phytopythium vexans. An interval from 0.01 μg/ml
to 10μg/ml was determined with Pythium dissotocum and P. vexans; while the
shortest interval (0.01-1μg/ml) was noted with P. nicotianae. In addition, present
findings recorded a difference in Pearson’s correlation index between growth
factors (medium, evaluation dates, mefenoxam concentration) and Oomycetes
mycelium growth using MA and CA methods. A statistical significance difference
(P<0.05), weas noted more in case MA method and was more expressed in case of
CA method.
Benfradj Najwa et al., 2022
2
1. Introduction
Oomycetes are a group of microorganisms that
shares microscopic hyphal morphology of
fungi and contains large number of
phytopathogens which cause disease in wide
crop. Differences between Oomycetes and true
fungi is that Oomycetes cell walls are
composed of β-1,3 and β-1,6 glucans, whereas
true fungi cell walls are composed of chitin
(Williams-Woodward and De Mott, 2013).
Fungi and Oomycetes mycelium grows out, on
a surface of a natural substratum, by an
expansion of spore or hypha results in a
collection of a fungal colony. After an isotropic
growth phase, hypha initiates random
branching, forming fractal tree-like colonies
(Fricker et al., 2007). As for fungi, Oomycetes
species growth is not easy to quantify because
these organisms do not grow as single cells, but
as hyphal filaments that cannot be quantified
by the usual enumeration techniques (Taniwaki
et al., 2006). Colonies shapes and surface
textures provide useful information to
determine species or to monitor growth state
(Matsuura, 1999). Chemical control is the most
used strategy to prevent Oomycetes diseases.
Mefenoxam, is the active ingredient of a
partially systemic fungicide largely used in the
management of diseases caused by various
Oomycetes (Olson et al., 2013). Mefenoxam
inhibit ribosomal RNA synthesis of Oomycetes
and thus suppressing the mycelial growth and
the sporulation of the pathogen. In vitro
evaluation of fungicide effect is the standard
bioassay used to determine chemistries that
influence growth of fungi and Oomycetes
pathogens; in this assay, fungicide is amended
into agar medium, and the response is recorded
after a specified time interval (Noel et al.,
2018). The fungal colony diameter is governed
by the assumption that colony diameter is a
good indicator of colony growth (Hendricks et
al., 2017). Thus, measurement of radial growth
is the most frequently method used as
parameters to evaluate Oomycetes growth in
this assay. Matsuura (1999) revealed that
colonies shapes and surface textures provide
useful information to determine the species or
to monitor the growth state; colonies
patterning looks to be highly sensitive to
environmental factors, however, there might
be underlying basic rules of pattern selection
common for any species. However,
Oomycetes species growth study are the
heterogeneity of growth environments. In fact,
morphological colonies of Oomycetes species
could have different aspects such as uniform,
stellate, radiate, petaloid/rosette and another
forma according to medium and growth
conditions. Furthermore, because of erratic
developmental changes in the contents of such
constituents such assays are generally not
recommendable for assessing fungal growth
inhibition (Broekaert et al., 1990). In addition,
due to the increasing availability of image-
capturing techniques (Falconer et al., 2010), an
interesting alternative is the capturing images
use because it’s easy and does not require
expensive equipment (De Ulzurrun et al.,
2015). The aim of this study was based on the
fact that Oomycetes species have different
colonies aspects, and that measurement of
diameter colonies could not be the most
suitable method to evaluate their growth state.
Thus, the objectives of this investigation were
to compare between calculated area (CA)
obtained by perpendicular diameter and
measured area (MA) recovered from image-
capturing techniques to detect difference in
colonies growth of some Oomycetes species.
2. Materials and methods
2.1 Isolates, culture conditions and
inoculum preparation
Seven Oomycetes species isolates were
investigated in this study. Two of these
isolates belong to the genus Phytophthora (P.
nicotianae, P. cryptogea), three belong to the
genus Pythium (P. ultimum, P.
aphanidermatum, P. dissotocum) and two
were identified as Phytopythium spp. (P.
Benfradj Najwa et al., 2022
3
mercuriale, P. vexans). These isolates were
recovered, between 2012 and 2013, from trunk
and soil of citrus trees infected by gummosis
and proved as the causative agents of this
disease in Tunisia (Boughalleb-M’Hamdi et
al., 2018; Benfradj et al., 2017). Initial cultures
of isolates were prepared by transferring stored
plugs of each one from conserved cultures in
sterile soil solution onto PDA (Potato-
Dextrose-Agar) medium plates. Before used,
isolates were cultured for 6 days at 25±1°C, in
darkness until mycelium covered the surface.
The main characteristics of the used isolates
are listed in Table (1).
Table 1: Characteristics of Oomycetes isolates obtained from infected citrus orchards by gummosis and
used in the present study.
Species
Localities
GenBank Accessions numbers
Hosts
P. nicotianae
Takelsa
KU248808
Thomson navel
P. cryptogea
Hawaria
KU248814
Clementine hernandina
P. ultimum
Takelsa
KU248786
Thomson navel
P. aphanidermatum
Menzel bouzalfa
KU248783
Clementine hernandina
P. dissotocum
Bouargoub
KU248782
Thomson navel
P. mercuriale
Takilsa
KU248804
Thomson navel
P. vexans
Bnikhaled
KU248800
Thomson navel
2.2 Preparation of fungicide concentrations
The evaluation of response of Oomycetes
species to mefenoxam (Ridomil Gold®EC,
96.2 % active ingredient, Novartis Crop
Protection, Inc.) was assessed using the
method of Pradhan et al. (2017). Thus, each
fungicide concentration was prepared via serial
dilutions in sterile distilled water in amber
glass bottles, mixed on a stirring plate at
medium speed for two minutes and used within
24 h. Minimum application rate (MAR) of
mefenoxam (10.425 μg/ml) is used according
to the estimation of Chen et al. (2001) who
assuming soil depth and soil bulk density to be
2 cm and 1.2 g/cm3, respectively. The first six
concentrations of mefenoxam were from MAR
× 103 to MAR × 10−3, in 10-fold dilutions and
the control (sterile distilled water) were used to
determine the benchmark dose (BMD). BMD
(≈100 μg/ml) was calculated using continuous
Hill model, by default parameters via
Benchmark Dose Software (BMDS, V.
2.7.0.4) (Flores and Garzon, 2013). BMD
values were used to calculate the ten
mefenoxam concentrations (BMD × 102, BMD
× 1010, BMD × 100.5, BMD, BMD × 10-0.5,
BMD × 10−1, BMD × 10-1.5, BMD × 10−2,
BMD × 10-2.5, BMD × 10−3) and prepared as a
10X stock solutions for the assays.
2.3 In vitro effect of mefenoxam doses on
Oomycetes mycelial growth
Mycelial growth of Oomycetes isolates was
evaluated onto PDA and CMA (Corn-Meal-
Agar) (Tulip Diagnostics, Goa, India)
medium. Used medium was prepared
according to the manufacturer’s instructions,
dispensed in number of flasks (250 ml) equal
to the number of treatments in each assay, and
autoclaved at 120°C, for 20min. The medium
was cooled down to 60°C, before adding each
solution of fungicide concentration. Each
medium-fungicide combination was stirred for
two minutes using magnetic stirrers and
poured into 9 cm-diam petri dishes containing
20 ml of each medium. After solidification,
agar plugs of each isolate (5 mm in diameter)
were placed on petri dishes center of medium
amended with each fungicide concentration.
Ten fungicide concentrations (1000, 100, 31.6,
10, 3.16, 1, 0.31, 0.1, 0.03 and 0.01 μg/ml) and
a control without fungicide (0 μg/ml) were
Benfradj Najwa et al., 2022
4
used for each experiment. Petri dishes were
sealed with parafilm and incubated at 25±1°C
in the dark, for 6 days.
2.4 Methods of growth measurement
Every 2, 4 and 6 days of incubation, isolates
growth was measured by calculated the colony
diameters area (CA) and measured the surface
area (MA) on PDA/CMA medium with or
without fungicide concentration. Diameter
area of each isolate was calculated from the
reverse side of petri dish (90mm) in
millimeters using a ruler. Mean diameter
growth for each isolate was measured by
averaging colony diameter measurements and
subtracting the plug diameter. For MA, isolates
images colony were scanned using CanoScan
8400 F (Canon, Melville, NY) and the whole
area of the colony was measured using the
software Statgraphics Plus 5.1.
2.5 Data analysis
For all experiments, three replications per
isolate–fungicide concentration combination
were carried, and each experiment was
repeated twice in the time. Methods of
measurement of Hendricks et al. (2017) were
used to determine if there is a difference in
analyzed results between CA and MA
measurements and whether these differences
changed the final conclusion made at the
treatment level. Thus, the average diameter of
each colony was converted to area of a circle
using the following formula:
With: DA: Diameter converted to area of circle;
D: average diameter of each colony.
Then, the ratio of mycelia growth inhibition of
each isolate by the fungicide were calculated
according to the formula:
For the comparison between the two methods
of evaluation, obtained data were subjected to
variance analysis (ANOVA) and multiple
comparisons of means was carried using
Student-Newman-Keuls test (P<0.05) as a post
hoc test (XLSTAT 2018.1. Software). In
addition, Principal component analysis (PCA)
at P < 0.05, with a Pearson correlation
coefficient at n = 1 were used to identify the
variation in Oomycetes species inhibition
ratio, in each evaluation method, across the
growth factors (STATISTICA 13.5.0.17).
3. Results
3.1 Evaluation of two methods of in-vitro
growth of Oomycetes species
Results of this study proved the growth
suppressive effect of mefenoxam on
Oomycetes species and the existence of
difference between two methods of growth
evaluation of these species. Both, using PDA
and CMA medium. The suppressive effect of
mefenoxam increase according to the
evaluation dates, in all Oomycetes species. No
difference between MA/CA methods was
noted at higher mefenoxam concentrations,
because of the complete inhibition of
Oomycetes mycelium growth (Ratio= 1).
However, MA method was more efficient than
CA method to determine subtle changes in
Oomycetes mycelia growth at variables
intervals of lower mefenoxam concentrations.
Intervals of variation between MA/CA
methods were influenced by Oomycetes
species growth factors (medium, dates,
concentrations).
Benfradj Najwa et al., 2022
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3.1.1 Case of Phytophthora species
Results recorded unequal interval of variation
between CA/MA methods using Phytophthora
species. For P. nicotianae, this interval was
between the concentrations 0.01 μg/ml and 1
μg/ml for both used medium. At this interval,
inhibition ratio was higher using PDA medium
(Figure 1a) then by CMA medium (Figure 1b)
(ANOVA, for Ratio MA (F)= 30.571/ for Ratio
CA (F)= 8.435, p < 0.004). Inhibition ratio of P.
nicotianae was ranged from 0.268 to 0.973
using MA method compared to an interval
from 0.128 to 0.829 by CA method. However,
MA/CA interval of variation of P. cryptogea
was largest then P. nicotianae one but
associated to used medium. In fact, using PDA
medium, this interval was ranged between the
concentrations 0.01 μg/ml and 31.6 μg/ml
(Figure 1a), while using CMA medium, it was
between 0.01 μg/ml and 100 μg/ml (Figure 1
b). At this interval, no difference was noted
between inhibition ratio of P. cryptogea in
PDA and CMA medium.
Figure 1: Comparison between calculated area (CA) and measured area (MA) of Phytophthora nicotianae and P.
cryptogea using PDA and CMA medium, amended with different concentrations of mefenoxam after 48 (a), 96 (b)
and 144h (c) of incubation at 25±1°C, in darkness.
Inhibition ratio of P. cryptogea was from 0.168
to 1 using MA method compared to an interval
from 0.011 to 0.901 by CA method. At these
intervals, 'Medium-Concentration-Day' effect
was highly significant between Ratio MA and
Ratio CA, having p-values of p<0.0001 in case
of P. nicotianae (for Ratio MA, F= 5.591/ for
Ratio CA, F= 5.270) and P. cryptogea (for Ratio
MA, F= 10.040/ for Ratio CA, F= 5.903).
3.1.2 Case of Pythium species
The largest interval of variation between
MA/CA methods was obtained with Pythium
aphanidermatum grown onto CMA medium
(0.01 μg/ml – 1000 μg/ml). The second
Benfradj Najwa et al., 2022
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interval of variation (0.01 μg/ml – 100 μg/ml)
was noted with Pythium ultimum using both
medium and in case of Pythium
aphanidermatum using PDA medium.
However, with Pythium dissotocum, the
interval of variation between MA/CA methods
was between 0.01 μg/ml and 10 μg/ml.
Inhibition ratio of P. ultimum was from 0.123
to 1 using MA method compared to an interval
from 0.083 to 0.857 by CA method. Inhibition
ratio of P. aphanidermatum was from 0. 213 to
1 using MA method compared to an interval
from 0.100 to 0.812 by CA method. However,
inhibition ratio of P. dissotocum was from 0.
179 to 0.980 using MA method compared to an
interval from 0.050 to 0.910 by CA method
(Figure 2). A high significant difference was
noted in ration between PDA and CMA
medium, using Pythium aphanidermatum
(ANOVA, Ratio MA (F)= 11.903/ for Ratio CA
(F)= 11.478, p<0.001). However, no
differences among medium used were
recorded neither for Pythium ultimum nor
Pythium dissotocum. Furthermore, 'Medium-
Concentrations-Day' effect was highly
significant between Ratio MA and Ratio CA,
having p-values of p<0.0001 in case of P.
ultimum (for Ratio MA, F= 7.235/ for Ratio CA,
F= 19.470), P. aphanidermatum (for Ratio MA,
F= 11.715/ for Ratio CA, F= 6.121) and P.
dissotocum (for Ratio MA, F= 10.463/ for Ratio
CA, F= 5.267).
Figure 2: Comparison between calculated area (CA) and measured area (MA) of Pythium aphanidermatum, P. ultimum
and P. dissotocum using PDA and CMA medium, amended with different concentrations of mefenoxam after 48 (a), 96
(b) and 144h (c) of incubation at 25±1°C, in darkness.
3.1.3 Case of Phytopythium species
For Phytopythium species, the interval of the
variation between MA and CA methods was
also variable according to the species and
depending on used medium. For Phytopythium
mercuriale, this interval was from 0.01 μg/ml
to 100 μg/ml using PDA medium and from
Benfradj Najwa et al., 2022
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0.01 μg/ml to 100μg/ml using CMA medium.
However, for Phytopythium vexans, the
interval was from 0.01μg/ml to 10μg/ml using
PDA medium and from 0.01 μg/ml to 31.6
μg/ml using CMA medium (Figure 3). A
significant difference of inhibition ratio noted
between PDA and CMA medium, using
Phytopythium mercuriale (ANOVA, Ratio MA
(F)= 22.695/ for Ratio CA (F)= 7.94146,
p<0.005).
Figure 3: Comparison between calculated area (CA) and measured area (MA) of Phytopythium mercuriale and P. vexans
using PDA and CMA medium, amended with different concentrations of mefenoxam after 48 (a), 96 (b) and 144h (c) of
incubation at 25±1°C, in darkness.
Phytopythium vexans revealed a significant
difference between the two-medium used just
in case of CA method (ANOVA, Ratio MA (F)=
5.362/ for Ratio CA (F)= 7.941, p<0.02).
Inhibition ratio of P. vexans was from 0. 146 to
1 using MA method compared to an interval
from 0.010 to 0.100 by CA method, while
inhibition ratio of P. mercuriale was from 0.
213 to 0.992 using MA method compared to an
interval from 0.106 to 0.887 by CA method.
Also, the effect 'Medium-Concentrations-Day'
was highly significant between Ratio MA and
Ratio CA, having p-values of p<0.0001 in case
of Phytopythium mercuriale (for Ratio MA, F=
6.251/ for Ratio CA, F= 6.803), and
Phytopythium vexans (for Ratio MA, F= 8.773/
for Ratio CA, F= 22.426).
3.2 Correlation between growth factors and
inhibition ratio of Oomycetes species
Regardless of the clear site separation detected
by the Principal Component Analysis in both
evaluation methods, a markedly differences
were noted. In MA method, the first
component (PCA1) explained 72.540 of the
sites variances and was mainly determined by
medium used. The second component (PCA2)
captured 10.575 of variance of site distribution
and was determined by the dates and the
Benfradj Najwa et al., 2022
8
mefenoxam concentrations. The same
components determination was obtained for
CA method. However, in this method, PCA1
explained 72.747% of sites variance, and
PCA2 captured 10.666%. Both using MA and
CA methods, growth factors and pathogens
inhibition ratio were grouped in the positive
end of PCA1. However, changes were
recorded in PCA2. In fact, for MA method
Phytophthora species, P. ultimum and P.
vexans were located in the positive end of
PCA2. However, P. aphanidermatum, P.
dissotocum and P. mercuriale were located on
the negative end of PCA2. In the other hand,
by CA method, all Phytophthora and
Phytopythium species, in addition with two
Pythium species (P. ultimum, P. dissotocum)
were located on the positive end of PCA1.
Grouped on the opposite PCA1 end was just
the pathogen P. aphanidermatum (Figure 4).
In comparing the matrix cubensis expression
profiles of MA and CA methods, a difference
in Pearson’s correlation index (r) among
inhibition ratio of Oomycetes species and
growth factors was noted. In MA method,
using medium the majority of Pearson’s
correlation index values were between -0.273
and -0.091, while values this correlation was
higher in case of CA method, ranged between
-0.091 and 0.091. However, using mefenoxam
difference between the degree of correlation
was noted just in case of P. nicotianae where
it was high in case of CA method (0.818-1)
than in case of MA method (0.273-0.455). In
addition, according to evaluation dates,
difference was noted in case of P. vexans with
higher correlation using CA method (0.091-
0.273) than using MA method (-0.091-0.051).
Figure 4: Difference among Principal components analysis (PCA) scatterplot between the grown factors and
inhibition ratio of Oomycetes species using MA and CA methods (P1: P. nicotianae; P2: P. cryptogea; P3: P.
ultimum; P4: P. aphanidermatum; P5: P. dissotocum; P6: P. mercuriale; P7: P. vexans). Black dots indicate average
measures of each factor.
A scatter plot matrix was generated by
performing the correlation matrix analysis
based on linear regression depicts the influence
of growth factors on Oomycetes growth in
each method of evaluation. Values with
statistical significance at the level P > 0.05
Benfradj Najwa et al., 2022
9
were noted more in case of MA method then in
case of CA method. In fact, with AM method,
P. nicotianae (-0.107) and P. aphanidermatum
(0.087) were found to be correlated to medium,
while P. mercuriale (0.109) shown to be
positively correlate with dates. However, by
CA method, just a one positive correlation was
found between dates and P. aphanidermatum
(0.100). In addition, Pearson’s correlation
index (r) has values with statistical
significative at the level P >0.01 for both
methods. In MA method, it is evident that
medium showed a good positive linear relation
(r = 0.87) with P. aphanidermatum, a negative
linear relation with P. nicotianae (-0.107), P.
cryptogea (-0.156), P. ultimum (-0.133), P.
vexans (-0.157). However, the correlation
values obtained for P. dissotocum (-0.015) and
P. mercuriale (-0.076) were insignificant. In
the other hand, using CA method just a
negative linear correlation was made between
medium and P. cryptogea (-0.238) and P.
ultimum (-0.164), while insignificant values
were noted with other species. In addition,
mefenoxam exhibited a notable positive linear
correlation with all Oomycetes species using
both methods. Interestingly, except P.
nicotianae (r=0.743), all Oomycetes species
shown a positive linear correlation (r>0.818).
Further, as shown, evaluation dates, exhibited
a moderately linear positive correlation with
all Oomycetes species (r>0.01) in case of MA
method. However, a in case of CA method, a
moderately linear positive correlation with all
Oomycetes species (r>0.01) were noted from
P. nicotianae, P. cryptogea, P. ultimum, P.
dissotocum, P mercuriale; while a low linear
positive correlation was noted with P. vexans
(r=0.047).
4. Discussion
The present work compares two methods of
evaluation of Oomycetes species mycelium
growth. Results of this study showed that
method of measured area was more efficient
then calculated area method to distinguish
differences in fungicide growth inhibition for
Oomycetes species. In fact, measured area was
more efficiency to distinguish subtle changes
in Oomycetes growth inhibition at low
concentrations of mefenoxam. Our results
agree with Hendricks et al. (2017) results who
revealed that calculated area and/or measured
area was adequate to distinguish significant
treatment effects of fungicide on fungal
growth, however MA was more sensitive. In
fact, in study of these authors noted that
analysis of area calculated from colony
diameter and measured area were adequate to
distinguish significant differences in fungicide
growth inhibition for Phyllosticta citricarpa at
lower fungicide concentrations. Difference
between the methods of evaluation found in
this study, could be due to that Oomycetes
hyphae are not grow completely straight and
produce extent. In fact, fungal growth and
direction occurs as the combined result of
various biological processes, such as the
ubiquity and ecological impact (Riquelme et
al., 1998), environmental stimuli (Graham,
1995). Much research was done on the ability
of hyphae to change growth direction
(tropisms) in response to external stimuli
(Riquelme et al., 1998). Measurement of the
growth rate-nutrient level relation for the
fungal strains is expected useful to understand
the diversity in the colony patterning under the
change of other environmental factors, such as
the stiffness of the substrate (Matsuura, 1999).
According to Riquelme et al. (1998), hyphae
elongate by apical growth, polarized and
growing tip is the likely place where the
growth directionality of a hypha is established.
In fact, the fungi ability to generate polarized
cells with a variety of shapes reflects a
temporal and spatial control over the polarity
axes formation (Riquelme et al., 2011). Angle
Benfradj Najwa et al., 2022
10
at which a new hypha branches relative to the
existing one depends on the species (Kamel et
al., 2009). Also, by growing and exploring, a
hypha can encounter another hypha and fuse
with it (anastomosis), as such changing the
fungal network shape and increasing the
nutrient cycle efficiency (Simonin et al., 2012).
Hyphal apex shape and diameter are results of
the relative rates of wall component synthesis
and rigidification (Graham, 1995). Oomycete
cell wall is composed of β-1,3, and β-1,6
glucans, and not of chitin (William et al.,
2010). Growing hyphae of fungi have
appreciable activities of chitinases and other
lytic enzymes (Graham, 1995). The
spitzenkörper position in the hyphal tip
determines the growth direction (De Ulzurruna
et al., 2017; Lopez- Franco and Bracker, 1996;
Girbardt, 1957). CA method limitations could
be explained by the fact that chitin content of
hyphae may change with age and growing
conditions (Matcham et al., 1985). Gooday
(1976) noted that difference in Oomycetes
colonies behavior may be explained by the fact
that their hyphae respond chemotropically to
nutrients such as sugars. In our study, the
composition and the nutrient in the two media
used are different. In fact, for Oomycetes
species, CMA media have more nutriment than
PDA media. According to Matsuura (1999),
hyphae were produced densely inside the
colony and oppositely at high nutrient level,
while at low nutrient levels colonies expand to
cover almost entire medium surface with far
less hyphal density. Measurement of fungal
strains growth rate-nutrient level relation is
expected useful to understand the diversity in
the colony patterning under the change of other
environmental factors, such as the stiffness of
the substrate (Olsson, 2001). According to
Trinci (1969), when comparisons are made
between species colony radial growth rate is
not a meaningful parameter of growth. In fact,
in his study, Trinci (1969) found that if the
environmental conditions within this
peripheral growth zone became unfavorable to
growth, the colony radial growth rate would
decrease; thus, this author affirm that colony
radial growth rate is not a reliable parameter of
specific growth rate in submerged culture in
studies where nutrient concentration is varied.
Results of our study showed differences
between colony morphology of Oomycetes
used. Also, Taniwaki et al. (2006) and
Schnürer (1993) noted differences in growth
patterns between different fungal species.
Olsson (2001) colony morphology and
medium could change due to fungal activities.
Mechanism behind this measurments efficient
is probably the ability of MA to distinguish
small changes in fungal growth inhibition. In
addition, one of the main advantages of the
image analysis method presented is that it is
completely automated does not require direct
interaction with the samples, allowing to
follow the entire fungi growth (De Ulzurrun et
al., 2015). The image gamma correction
applied is important to enhance the contrast
between the object of interest and the
background, because the colony area of the is
completely isolated from the remaining image
(Da Silva et al., 2017). By using proper masks
on the original images, it could be possible to
study local behavior within the colony, such as
the difference in growth behavior and
morphology between the central and the
peripheral region where hyphae avoid contact
and generate less branches and fusions
(Riquelme et al., 2011). However, this method
of measurement needs a high contrast picture
of the colonies. In the other hand, although CA
method shown to be an easy and good
measurement approach, only horizontal
growth is considered in this method. CA
method doesn’t take in consideration the
pathogen vertical growth or the density
increase in the Petri dish. Also, a drawback of
using two-dimensional measures such as area
Benfradj Najwa et al., 2022
11
or diameter is that the third colony density
dimension is not taken into consideration
(Taniwaki et al., 2006). In addition,
interactions between fungi and their
environment are often neglected (De
Ulzurruna et al., 2017). A difference was also
noted between MA/CA methods correlation
matrices. Overall, using both methods a
negative correlation was mentioned between
pathogens and medium used. However, a
positive correlation was noted between
pathogens and mefenoxam and evaluation
dates. Results classified the degree correlation
of factors as high in CA method compared to
MA one. This could be explaining by the low
growth rate obtained in case this method
compared to MA one. According to Miyashira
et al. (2010) and Loeck et al. (2004), the low
growth rate has been considered as a limiting
factor regarding several experimental
analyses.
5. Conclusion
The present work compares two methods of
evaluation of Oomycetes species mycelium
growth. Results of this study showed that
method of measured area was more efficient
then calculated area method to distinguish
differences in fungicide growth inhibition for
Oomycetes species. In fact, measured area was
more efficiency to distinguish subtle changes
in Oomycetes growth inhibition at low
concentrations of mefenoxam.
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