Introduction
The fungus C. falcatum is the causal agent of
sugarcane red rot, one of the most destructive diseases that affects the crop (Khan
et al. 2011; Bharti et al. 2012; Sharma and Tamta 2015). Widely disseminated across all continents, sucrose yield losses of
50–70% have been reported in infected stems (Santiago and
Rossetto 2008). Earliest red
rot epidemics were atributed to attach of Diatrea saccaralis, but the
fungus was no longer 100% associated with Diatrea saccharalis, more
aggressive variants do not need the hole of the insect to penetrate the stalk. With the absence of burned, the pathogen survives in the
soil and crop residues after harvest and with each harvest the incidence and
severity increases (Viswanathan 2010), it has become a very harmful
disease to sugarcane crops in countries like Brazil and India (Viswanathan et
al. 2020a).
The fungal pathogen exhibits
enormous variation under fields conditions and the pathogenics variants emerge
regularly in tune with deployment of new host varieties for cultivation making
the resistant to susceptible referred as “varietal breakdown” (Viswanathan et
al. 2020b). So, already many sugarcane varieties were replaced due to their
breakdown to a new pathogenic strain, so chemical control may be more
one possibility in the management of red rot in sugarcane crop. Given the losses caused by the disease, existence of pathogenic
variation in C. falcatum and the emergence of new virulent pathotypes
were documented over the decades (Viswanathan 2010; Viswanathan et al.
2020b).
Among the management strategies
used, fungicides are an important supplementary tool in controlling different
diseases (Nene and Thapliyal 1993). However, in the case of sugarcane red rot,
information on the sensitivity of various C. falcatum isolates to
different fungicides remains scarce, largely due to the lack of a practical and
efficient method to conduct this assessment. Conventional techniques for
evaluating the fungitoxicity of active ingredients in a fungicide-enriched
growth medium by inhibiting mycelial growth and/or conidial germination are
costly, laborious, time-consuming, require a significant amount of laboratory
space to accommodate a large number of plates, cannot identify intermediate
sensitivity to fungicides and preclude automated data collection (Rampersad and
Teelucksingh 2012; Promega Corporation 2019).
An alternative strategy for
assessing the fungitoxicity of active ingredients is resazurin, a stable,
nontoxic water-soluble dye used as an indicator in oxidation-reduction
reactions. Resazurin has been used to measure cell proliferation, as well as
the viability and cytotoxicity of different cell types in medical research (Invitrogen
Molecular Probes 2021; ThermoFisher Scientific 2021). As such, the present
study aimed to (i) adjust a colorimetric method for assessing the sensitivity
of C. falcatum isolates to the main fungicide chemical groups
strobilurin, triazole, benzimidazole, isophthalonitrile, and dithiocarbamate
and (ii) evaluate the fungitoxicity and sensitivity of the different C.
falcatum isolates of the active ingredients tested.
Materials and Methods
Preparation of the conidial
suspension
Eighteen fungal isolates were obtained from monoconidial C. falcatum cultures
belonging to the microorganism collection of the Plant Pathology Laboratory at
the School of Agronomy of the Federal University of Goias, collected in the
state of Goias in the 2017 growing season. For the production of conidia, the
isolates were cultivated in Petri dishes containing potato dextrose agar (PDA)
medium for 20 days at 28–30°C, under constant darkness.
In order to determine the optimal
conidia concentration to test the sensitivity to fungicides of 18 C. falcatum
isolates, suspensions were prepared at initial concentrations of 104,
105 and 106 conidia mL-1 for one C.
falcatum isolate (CFF 12). The
conidial suspension (60 µL), 140 µL of Czapek Dox Broth medium + 0.05% agar and 10 µL
of 0.02% resazurin were deposited into a clear flat bottom polystyrene
microplate. Each conidial suspension concentration was deposited in three
repetitions (microplate wells). The negative control used was 60 µL
sterile water without conidia, also in three repetitions. The microplate was
incubated in a biochemical oxygen demand (BOD) incubator at 28–30°C, under
constant darkness. In order to determine the ideal incubation period, the
resazurin reduction percentage was evaluated after 48 and 72 h of incubation.
Fungicide sensitivity testing
The following fungicides were
tested: Amistar® WG 500 g kg-1 of Azoxystrobin, obtained
from Syngenta), Tilt® (250 g L-1
of propiconazole, Syngenta), Carbendazim (97%, Sigma-Aldrich), Manzate 800 (800
g kg-1 of mancozeb, UPL), Bravonil® 720 (720 g L-1
of chlorothalonil, Syngenta) and Cercobin 700 WP (700 g kg-1 of
thiophanate-methyl, Iharabras). Each fungicide was precisely weighed and
dissolved in Czapek
Dox Broth medium + 0.05% agar (used as a dispersing
agent) in serial dilutions. The active ingredient concentrations used were 0;
0.01; 0.1; 0.5; 1; 5; 10 and 50 µg mL-1.
Suspensions with a concentration
105 conidia mL-1 were prepared for each of the 18
isolates. Each well received 60 µL of conidial suspension, 140 µL
of Czapek Dox Broth medium + 0.05% agar,
active ingredients at the doses previously mentioned and 10 µL of 0.02%
resazurin. For strobilurin, 10 µL of
salicylhydroxamic acid (SHAM) was added to the well to suppress the alternative
oxidase pathway (Ma et al. 2006). Active ingredient doses were tested in
three wells for each isolate. The
microplates were sealed and incubated in a BOD incubator at 28–30°C under
constant darkness. The resazurin reduction percentage was evaluated after 48 h of
incubation.
Absorbance of the microplate
wells was read in an Epoch microplate spectrophotometer (Biotek, Vermont,
United States) after 48 h of incubation, at wavelengths of 570 nm and 600 nm,
with the aid of GENE 5 software, version 2.0. The resazurin reduction
percentage was estimated using the formula provided by the manufacturer of
alamarBlue® reagent (Cox et al. 2009).
Data analysis
Data were analyzed in R software
version 3.6.2. For each dose of active ingredient (dose y), the growth
inhibition percentage (GIP) of the isolate was calculated in relation to dose
zero (dose 0) using the estimated resazurin reduction percentage, which
indirectly measures initial C. falcatum growth. For each active
ingredient, the GIP as a function of dose and isolates was submitted to
analysis of variance, using the ExpDes package of R software. The nls
and nlstools packages were used for nonlinear regression adjustment
based on the model proposed on Michaelis–Menten model, where GIP is a function
of the active ingredient dose.
Results
Adjustment of spore concentration and incubation time
Preliminary tests to adjust the
spore concentration and incubation time were assessed by analysis of variance
and Tukey’s test (α = 0.01). A concentration of 105 conidia mL-1
provided the greatest resazurin reduction, with no significant difference
between incubation times of 48 and 72 h, confirming the stability of the dye after
48 h of incubation.
Sensitivity of C. falcatum isolates to fungicides
For each active ingredient, the C.
falcatum GIP as a function of dose and isolates was submitted to analysis
of variance, with no significant difference in GIP between C. falcatum isolates
(α = 0.01) (data
not shown). The regression curve that expresses the C. falcatum GIP as a
function of dose, based on the model proposed by (Michaelis and Menten 1913),
was adjusted for each active ingredient. Thus, all the regression curves were
significant at 0.1% according to the F-test. The regression equations,
coefficients of determination (R2) and doses that inhibited 50% of
growth (EC50) for each active ingredient are described in Table 1.
The
effects of the active ingredients varied in relation to fungitoxicity to C.
falcatum. The sensitivity of C. falcatum to the molecules tested can
be ranked in descending order based on EC50, as follows: azoxystrobin, propiconazole, carbendazim, mancozeb, chlorothalonil and
thiophanate-methyl.
Comparison of the fungitoxicity of the active ingredients is shown in Fig. 1
and 2.
%2083-87_files/image002.png)
Fig. 1: Visualization of Colletotrichum falcatum growth
inhibition by resazurin reduction as a function of fungicide doses.
%2083-87_files/image004.png)
Fig. 2:
Comparative regression curves of Colletotrichum falcatum
growth inhibition as a function of fungicide fungitoxicity. CI: confidence
interval
Table 1: Regression equation,
coefficients R2 e EC50
|
Active Ingredient |
Equation |
R2 |
EC50 (µg.mL-1) |
|
Azoxystrobin |
GIP = -0.13 + 85.63. Dose /
(0.0069 + Dose) |
0.994 |
0.0097 |
|
Propiconazole |
GIP = -1.41 + 82.87. Dose /
(0.0542 + Dose) |
0.989 |
0.0885 |
|
Carbendazim |
GIP = -2.26 + 89.78. Dose /
(0.1916 + Dose) |
0.931 |
0.2668 |
|
Mancozebe |
GIP = 2.84 + 86.09. Dose /
(0.3161 + Dose) |
0.954 |
0.3829 |
|
Chlorothalonil |
GIP = -3.24 + 93.56. Dose /
(0.3281 + Dose) |
0.956 |
0.4332 |
|
Thiofanate methil |
GIP = 2.28 + 83.92. Dose /
(0.8519 + Dose) |
0.987 |
1.1230 |
Discussion
With respect to the spore concentration that optimizes resazurin reduction, the present study demonstrated that 105 conidia mL-1 provided the best results after 48 h of incubation. Resazurin sensitivity to spore density is dependent on the fungal species studied and should be determined empirically for studies involving dye reduction in response to the fungitoxicity of pesticides to phytopathogenic fungi (Cox et al. 2009). Larson et al. (1997) analyzed the viability of corneal endothelial cells and Pelloux-Prayer et al. (1998) the viability of Botrytis cinereal conidia, with both studies reporting that densities lower than 104 cells mL-1 required longer incubation times to reduce resazurin, whereas more than 105 cells mL-1 produced inconsistent results.
Larson et al. (1997) also found that high cell densities and
prolonged incubation times cause resazurin to reach an undesirable plateau
because the (blue) dye is reduced to resorufin (pink which, in turn, is reduced
to hydroresorufin (colorless). The reduction percentage for resorufin is higher
than that of hydroresorufin (Rampersad 2011).
In
regard to the incubation times tested, resazurin reduction stabilized between 48
and 72 h. This indicates that the dye reached its reduction limit at 48 h,
which was the time established for testing to assess C. falcatum sensitivity
to fungicides. Prolonged incubation times compromise resazurin reduction, as
reported by Larson et al. (1997).
There was no significant difference in growth inhibition, estimated by the resazurin reduction percentage (α = 0.01), between isolates for any of the active ingredients tested. As such, the C. falcatum isolates studied showed equal sensitivity to azoxystrobin, propiconazole, carbendazim, mancozeb, chlorothalonil and thiophanate-methyl.
Ghazanfar et al. (2017)
reported that Tilt® (250 g.L-1 of propiconazole) was more
efficient than mancozebe at inhibiting the mycelial
growth of the fungus and Nenhuma fonte bibliográfica
especificada. It was found more effective at
controlling sugarcane red rot.
In the present study, the active
ingredient propiconazole was the second most toxic to the pathogen (Fig. 1 and
2). In research on other chemical molecules, Nikhil and Sahu
(2014) observed that carbendazim was more effective
than propiconazole at inhibiting mycelial growth, whereas Abbas
et al. (2016) reported that mancozebe was most
effective, followed by propiconazole, and found that benomyl, tebuconazole and
mancozeb were more efficient than propiconazole. Although benomyl and tebuconazole
were not tested here, carbendazim was only less toxic to C. falcatum than
azoxystrobin and propiconazole, with mancozeb ranked fourth in terms of
fungitoxicity to the molecules tested (Fig. 1 and 2).
Studies that evaluate the
sensitivity of C. falcatum to different fungicides measure the effect of
different products on the mycelial growth of the fungus and/or conidial
germination in a culture medium, generally with doses above 5 µg mL-1.
However, a number of investigations do not indicate whether the dose refers to
active ingredients or commercial products (Bharti et al. 2014; Ghazanfar
et al. 2017). In our study, doses greater than 5 µg mL-1 were too high, which
precluded determining the fungitoxicity of azoxystrobin, propiconazole,
carbendazim, mancozeb, chlorothalonil, and thiophanate-methyl (Fig. 1).
Although the aforementioned studies evaluated the sensitivity of the pathogen
to different fungicides, none provided data on the active ingredient dose (EC50)
that inhibited 50% of mycelial growth and/or 50% of conidial germination, which
precluded comparison with the EC50 values found here (Nikhil and
Sahu 2014; Bharti et al. 2014; Abbas et al. 2016).
EC50
has been estimated for other species of the genus Colletotrichum.
Analysis of the conidial germination of C. gloeosporioides and C.
capsica isolates showed EC50 values from 0.009 to 0.091 µg
mL-1 for both species (Li et al. 2005). In our study, the EC50
of azoxystrobin for C. falcatum, calculated based on the estimated resazurin reduction percentage, was 0.0097 µg mL-1,
within the range described above (Table 1). The EC50
values for the active ingredients propiconazole and thiophanate-methyl
were estimated by the mycelial growth of C. cereale
isolates and varied from 0.025 to 0.35 µg mL-1 and 0.14 to
2.3 µg mL-1, respectively (Wong and Midland 2007; Wong et
al. 2008). In the present study, the EC50 values of
propiconazole and thiophanate-methyl for C. falcatum were 0.0885 µg mL-1 and
1.123 µg mL-1, respectively, similar to those reported for C.
cereale.
The
EC50 values of carbendazim and mancozeb for C. acutatum isolates
estimated, respectively, by the inhibition of mycelial growth and conidial
germination, were 0.1946 µg mL-1 for carbendazim and between
0.24 and 0.85 µg mL-1 for
mancozeb (Cai et al. 2008; Gao et al. 2017). For C. falcatum, our
study demonstrated EC50 values of 0.2668 µg mL-1
for carbendazim and 0.3829 µg mL-1 for mancozeb, calculated
based on the estimated resazurin reduction percentage, similar to the values
reported for C. acutatum.
The EC50
of chlorothalonil was estimated for C. truncatum and C.
gloeosporioides by mycelial growth inhibition, with values from 0.23 to
1.14 µg mL-1 and 0.01 to 0.95 µg mL-1,
respectively (Rampersad and Teelucksingh 2012). For C. falcatum, our
study recorded an EC50 of 0.4332 µg mL-1
for chlorothalonil, calculated via the estimated resazurin reduction percentage,
which is within the ranges reported for C. truncatum and C.
gloeosporioides.
The
aforementioned studies found similar EC50 values for the active
ingredients in question for species from the genus Colletotrichum to
those recorded to C. falcatum. Some of the studies calculated the EC50
for each isolate used and established a range for that dose. However, when
isolates exhibit similar sensitivity to fungicides, as observed for the C.
falcatum isolates tested here, EC50 is estimated for a set of
fungal isolates. Application of the fungicides showed a reduction in red rot incidence over
control but the extent of reduction varied considerably (10.6–39.4% reduction) across the treatments over the 2 years.
Overall, among the seven fungicides evaluated, sett treatment with two
fungicides (Tebuconazole + Trifloxystrobin and Thiophanate methyl)
consistently resulted in > 30% reduction in red rot incidence
over control in both years along with
significantly higher NMC over control (Joshi 2021).
Resazurin has been used
successfully in active ingredient fungitoxicity tests in several agronomic
studies. Research has reported a high correlation between the results obtained
with the traditional method and the resazurin-based assay (Cox et al.
2009; Vega et al. 2012). In
summary, we found that the resazurin method is a faster, low-cost alternative
to mycelial growth assays for C. falcatum. This assay may be a more
reliable indicator of fungicide resistance because it uses spores instead of
mycelia (Hu et al. 2007). Furthermore, it can provide sugarcane growers
with needed information about the sensitivity of isolates collected from their
orchards in a more cost-effective manner. The technology also adds simplicity
to the detection of resistance, especially when quantification is not desired,
and results can be taken visually.
Conclusion
The colorimetric method used to
assess the fungitoxicity of active ingredients to C. falcatum, combined
with resazurin, is fast, practical, efficient and viable for application in
large sets of isolates. All
the C. falcatum isolates studied exhibited equal sensitivity to
azoxystrobin, propiconazole, carbendazim, mancozeb, chlorothalonil and
thiophanate-methyl. The active ingredients displayed different levels of
toxicity to C. falcatum, ranked in descending order as follows:
azoxystrobin, propiconazole, carbendazim, mancozeb, chlorothalonil and
thiophanate-methyl.
Acknowledgements
The authors are grateful to the
EMBRAPA for providing necessary facilities to use the microplate
spectrophotometer at the institution. We would like to thank CNPq and Capes for
financial support. This work would not have been possible without equipment
provides for FAPEG.
Author
Contributions
RCM
and MGC planned the experiments, RCM, MGS and TMRO executed the tests, GAR,
RCM, VDD and RCF made the write up and statistically analyzed the data.
Conflict
of Interest
All
authors declare no conflict of interest
Data
Availability
Data
presented in this study will be available on a fair request to the
corresponding author.
Ethics
Approval
Not
applicable in this paper.
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