IntRasPecIfIc vaRIabIlIty In gRowth and In vItRo  
PRoductIon of Plant cell wall-degRadIng enzymes  
among aRgentInean Isolates of colletotRIchum  
gRamInIcola, a maIze Pathogen  
vaRIabIlIdad IntRaesPecífIca en el cRecImIento y la PRoduccIón In vItRo  
de enzImas degRadadoRas de PaRed celulaR vegetal entRe aIslamIentos  
aRgentInos de colletotRIchum gRamInIcola, un Patógeno de maíz  
1
1
1
María del P. Núñez , Laura N. Levin * & Isabel E. Cinto  
Resumen  
Introducción y objetivos: Colletotrichum graminicola (Glomerellaceae, Glomerellales)  
el agente causal de la antracnosis del maíz es dependiente de la actividad de  
enzimas degradadoras de la pared celular vegetal, para penetrar en su hospedante.  
La producción de estas enzimas se considera un factor de virulencia. El objetivo del  
presente trabajo fue investigar si existe variabilidad entre aislamientos en la capacidad  
de crecimiento y producción in vitro de diversas enzimas involucradas en la degradación  
de pared celular vegetal.  
1
. Laboratorio de Micología  
Experimental, DBBE-FCEN-UBA,  
INMIBO-CONICET, Buenos Aires,  
Argentina  
*isa.cinto@gmail.com  
M&M:SeevaluólahabilidaddeochoaislamientosdeC.graminícolaparacrecerysintetizar  
enzimas con actividad poligalacturonasa, polimetilgalacturonasa, β-glucosidasa y  
lacasa en cultivos líquidos utilizando dos medios de diferente composición.  
Resultados: La producción de poligalacturonasa, polimetilgalacturonasa y β-glucosidasa  
difirió marcadamente entre aislamientos y medios de cultivo. Se detectó actividad lacasa  
sólo en tres de los aislamientos. Los máximos títulos enzimáticos obtenidos fueron  
respectivamente de 250, 280, 45 y 63 U/l. La variabilidad intraespecífica registrada  
en la producción enzimática es consistente con la alta variabilidad intraespecífica  
observada a nivel genético cuando se evaluaron marcadores moleculares ISSR.  
Conclusiones: Los aislamientos de C. graminicola investigados mostraron notables  
diferencias en cuanto a la producción de enzimas degradadoras de pared celular  
vegetal, no asociadas a su capacidad de crecimiento. Esto indica una importante  
variabilidad intraespecífica que debería tenerse en cuenta al seleccionar un método  
para combatir a este patógeno.  
Citar este artículo  
NÚÑEZ, M. DEL P., L. N. LEVIN & I. E.  
CINTO.2023.Intraspecificvariability  
in growth and in vitro production of  
plant cell wall-degrading enzymes  
among Argentinean isolates of  
Colletotrichum graminicola,  
maize pathogen. Bol. Soc. Argent.  
Bot. 58: 187-194.  
a
DOI: https://doi.  
org/10.31055/1851.2372.v58.  
n2.39049  
PalabRas claves  
Antracnosis, Colletotrichum graminicola, enzimas degradadoras de pared celular, maíz.  
summaRy  
Background and aims: Colletotrichum graminicola (Glomerellaceae, Glomerellales),  
the causal agent of maize (Zea mays) anthracnose, as many other fungal pathogens,  
relies on its battery of cell wall degrading enzymes (CWDEs) to make its way through  
the cell walls of the host, and thus the production of these enzymes is considered a  
virulence factor. The aim of this work was to investigate if there is intraspecific variability  
in growth and in vitro production of several extracellular CWDEs among Argentinean  
fungal isolates of C. graminicola.  
M&M: Eight isolates of C. graminicola were tested in vitro to evaluate growth capacity and  
polygalacturonase, polymethylgalacturonase, β-glucosidase and laccase production,  
using two different liquid culture media.  
Results: Polygalacturonase, polymethylgalacturonase and β-glucosidase production  
greatly varied among isolates and culture media. Laccase activity was detected only in  
three isolates. Utmost enzymatic titres attained were respectively 250, 280, 45 and 63  
U/l. The observed intraspecific variability in CWDEs in vitro production is consistent with  
the high variability found at genetic level when assessing ISSR markers.  
Conclusions: The isolates of C. graminicola evaluated showed notable differences in  
CWDEs production, not associated with a differential growth. This indicates a large  
intraspecific variability, which might be considered when choosing a method to deal  
with this pathogen.  
Recibido: 19 Oct 2022  
Aceptado: 24 Abr 2023  
Publicado en línea: 1 Jun 2023  
Publicado impreso: 30 Jun 2023  
Editor: Leopoldo Iannone  
Key woRds  
Anthracnose, cell wall degrading enzymes, Colletotrichum graminicola, maize.  
ISSN versión impresa 0373-580X  
ISSN versión on-line 1851-2372  
187  
Bol. Soc. Argent. Bot. 58 (2) 2023  
IntRoductIon  
to obtain nutrients from plant polymers. These  
enzymes include ligninases, pectinases, cellulases,  
The prevalence of infections in crops has hemicellulases and various other hydrolases  
increased considerably lately, affecting harvests that target the cell wall polymers (Kubicek et  
around the world. Among all biotic threats, fungi al., 2014). The amount and variety of enzymes  
and oomycetes are the ones that pose the greatest released depends on the pathogen’s lifestyle.  
risk to global food security. Diverse factors are Most necrotrophs discharge copious amounts  
involved in this phenomenon, which include of enzymes, in an attempt to degrade as much  
changing climate conditions and agricultural as possible before the plant is able to stand  
practices. Higher temperatures in mild winters an effective defence; while biotrophs secrete  
allow pathogens survival and expansion to new relatively few CWDEs, thus operating by stealth  
areas, and accelerate their life cycle (Bebber and minimizing host damage. This difference  
&
Gurr, 2015). Many of these tropical fungal in enzyme production is also reflected in the  
pathogens are expanding to subtropical areas, pathogen’s genome: biotrophs encode less CWDEs  
setting new threats to agricultural production. when compared to other necrotrophic pathogens  
The Ascomycete Colletotrichum graminicola (Wang et al., 2022). C. graminicola, like most  
Ces.) Wils. (Glomerellaceae, Glomerellales) is Colletotrichum spp. species, has a hemibiotrophic  
(
recognized in Argentina as an important pathogen lifestyle. During the first stages of the disease, it  
in maize (Zea mays L.), causing anthracnose lives as a biotrophic pathogen, spreading through  
and stalk rot (Díaz et al., 2012). This disease is the plant tissue while inflicting minimum damage  
relatively new in the country: during the 1970’s to the host, before shifting to a more aggressive  
was considered only as a sorghum pathogen necrotrophic stage, where extended areas of the  
(
Fernández-Valiela, 1979). C. graminicola, host tissue are killed (Münch et al., 2008; Torres  
as initially described by Wilson, was a single et al., 2016). CWDEs are required for virulence  
cosmopolitan species with a very wide host range in many phytopathogenic fungi (Wijesundera  
among grasses. However, improved understanding et al., 1989; Paccanaro et al., 2017; Ma et al.,  
of C. graminicola taxonomy has confirmed that 2019). Several studies have demonstrated the  
the fungus is specific to Z. mays (Belisário et al., importance of pectinolytic enzymes in plant  
2
022). According to Dean et al. (2012), virtually invasion and symptom development (Reignault  
every crop grown is susceptible to at least one et al., 2008; Armesto et al., 2019). Ramos et al.  
species of Colletotrichum spp. These fungi cause (2010) showed that the disparities in the production  
anthracnose spots and blights on aerial plant parts of polymethylgalacturonase, polygalacturonase  
and post-harvest rots, generating major losses to and pectin lyase activities by C. truncatum isolates  
economically important crops. Anthracnose in (the causal agent of soybean anthracnose) were not  
maize causes important yield losses, because it related with fungal growth or geographical origin,  
prevents the kernel´s development or damages the but instead were associated with differences in  
stems before harvest. No-till farming techniques virulence among isolates. Ligninolytic oxidative  
without a proper crop rotation have also increased enzymes such as laccase also play an important role  
the incidence and severity of maize anthracnose in tissue maceration, as lignin is a complex aromatic  
in both North and South America (da Costa et al., polymer that hinders enzyme diffusion and can alter  
2
014). Estimates of yield reductions are as high as the extent of the enzymatic degradation of other  
1
0 to 20% worldwide (Belisário et al., 2022). In cell wall polymers. These enzymes also show a  
nearby countries such as Brazil, this disease has protective effect on the phytopathogen, as they are  
been responsible for most yield losses of maize able to detoxify phytoalexins and other phenolic  
(
Sukno et al., 2008). C. graminicola has been compounds that are involved in host’s defence  
reported as one of the most frequently found fungal mechanism (Vetchinkina et al., 2022).  
pathogens in Argentina recently; specially affecting  
Considering that maize anthracnose is a relatively  
second season maize crops (De Rossi et al., 2016). new disease in Argentina, a better understanding of  
Cell wall degrading enzymes (CWDEs) allowed the physiological behaviour of local isolates of  
host penetration and colonization, and are utilized this fungal pathogen is key to prevent and/or treat  
188  
M. del P. Núñez et al. - Colletotrichum graminicola: producción de enzimas degradadoras de pared celular vegetal  
the disease, and will contribute to an ecologically Agar (MEA). Incubation was carried out at 23  
sustainable integrated management. In this study, ºC under stationary conditions. Cultures were  
we analysed the intraspecific variability of eight harvested at different incubation periods, filtered  
native isolates of C. graminicola (obtained from through a filter paper using a Buchner funnel and  
symptomatic maize plants collected in different dried for 24 h at 70 ºC. Dry weight of mycelium  
production sites inArgentina), related to growth and was then determined. The culture supernatants  
activity of different extracellular CWDEs putatively were used as enzyme sources.  
involved in fungal penetration and colonization.  
Enzyme assays  
Polymethylgalacturonase (PMG) and  
mateRIals and methods  
polygalacturonase (PG) (endo and exo activities)  
were assayed using 0.1% apple pectin or  
polygalacturonic acid respectively, in 50 mM  
Fungi  
The isolates of C. graminicola were obtained sodium acetate buffer (pH 4.8) at 30 ºC. Liberated  
from lesions of symptomatic maize plants. They reducing sugars were quantified by the Somogyi-  
were collected from eight Argentinean localities in Nelson method (Somogyi, 1952). One unit of  
the provinces of Buenos Aires [CG 4(6) (Arrecifes enzymatic activity was defined as the amount of  
3
1
1
4° 04′ 00″ S, 60° 07′ 00″ W), CG20 (Rojas 34° enzyme releasing 1 µmol of galacturonic acid  
1′ 00″ S, 60° 44′ 00″ W), CG21 (Bolivar 36° per min. Laccase activity was measured using  
5′ S, 61° 06′ W) CG22(1) CG22(2), CG23(4) 2,6-dimetoxyphenol (DMP) 5 mM in 0.1 M sodium  
and CG27 (Pergamino 33° 53′ 01″ S, 60° 34′ 01″ acetate buffer (pH 3.6) at 30 ºC (Paszczynski  
W)] and Santa Fe [CG25(5) (Caseros 33° 03′ 00″ & Crawford, 1991). Oxidation of DMP was  
S, 61° 10′ 00″ W)]. The isolates are deposited determined by the increase in absorbance at 469 nm  
−
1
at the Phytopathology Department, Agronomy (ε469 = 27 mM·cm ). β-glucosidase was determined  
Faculty, University of Buenos Aires, Buenos Aires, in 50 mM sodium acetate buffer (pH 4.8) at 30  
Argentina.  
℃, by measuring p-nitrophenol released from  
.02% p-nitrophenyl β-D-glucopyranoside (Wood  
& Bhat, 1988). Enzyme activities were expressed  
0
Culture media  
Pectin culture (PEC) and Galhaup (GAL) media in International Units (U) as the amount of enzyme  
were used. Both media shared a basal medium required to release 1 μmol of product in 1 min.  
consisting of: MgSO .7H O, 0.5 g; HK PO , 0.6 g;  
4
2
2
4
H KPO , 0.5 g; CuSO .5H O, 0.4 mg; MnCl .4H O Statistical analysis  
2
4
4
2
2
2
0
.09 mg; H BO , 0.07 mg; NaMoO .2H O, 0.02  
The data presented in graphics was analysed  
3
3
4
2
mg; ZnCl , 2.5 mg; ZnCl 2.5 mg; FeCl 1.0 mg; using Graph Pad Prism 9.4 software. Two way  
2
2
3
biotin 5 μg; thiamine 100 μg; distilled water up ANOVA tests were performed and the differences  
to 1000 ml. For PEC, pectin from apple (10 g/l) between treatments were compared by Tukey’s  
and asparagine monohydrate (4 g/l) were added multiple comparison tests (p<0.05).  
as carbon and nitrogen sources respectively, and  
additionally 0.5 g/l of MgSO and 0.4 mg/l of  
4
CuSO were supplemented to the basal culture Results and dIscussIon  
4
medium (Ramos et al., 2010). For GAL, glucose  
(40 g/l) was used as carbon source, and 5 g/l of  
Kinetics of growth (Fig. 1) and in vitro  
yeast extract, 5 g/l peptone, 0.25 g/l CuSO and 1 production of several extracellular CWDEs (Fig.  
4
g/l SO Mg were added to the basal culture medium 2) by eight fungal isolates of C. graminicola was  
4
(
modified from Galhaup et al., 2002).  
characterized in two liquid culture media (PEC  
and GAL) based on pectin and glucose as carbon  
sources, respectively. A two-way ANOVA analysis  
Culture conditions  
Erlenmeyer flasks (125 ml) with 25 ml of revealed a significant effect of isolate, medium  
medium were inoculated with one agar plug (0.25 and interaction between factors (p<0.05) for each  
2
cm ) cut out from a colony grown on Malt Extract enzyme studied.  
189  
Bol. Soc. Argent. Bot. 58 (2) 2023  
Fig. 1. Kinetics of growth of C. graminicola isolates. A: in PEC media. B: in GAL media. Each point  
represents the mean of three replicate experiments, error bars denote SEM.  
All isolates were able to grow in both media, repression by the abundant reducing sugars detected  
showing maxima between 8 and 10 days. More in GAL medium during the first days of incubation  
biomass was obtained in GAL (Fig. 1B) than in (data not shown). PMG activity was not detected  
PEC medium (Fig. 1A) (approx. 21 mg/ml vs. 3 in isolate CG22(1). Most isolates exhibited the  
mg/ml), probably due to the more easily assimilable highest activity after the day of maximum growth  
carbon source. Values of biomass yield attained in (up to 0.28 U/ml), on the contrary in C. truncatum,  
PEC medium were similar to those obtained when the peak of PMG activity preceded this day (Ramos  
culturing C. truncatum in the same medium (Ramos et al., 2010). All isolates showed PG activity in  
et al., 2010).  
GAL medium, while only five of them showed  
No pectinolytic activity production was detected PG activity in PEC medium (Fig. 2B). The highest  
inPECmediumforsomestrains(Fig. 2A-B).Among activity (0.25 U/ml), was reached in GAL medium  
CWDEs, those with pectinolytic activity have been by isolate CG22(2). Most C. graminicola isolates  
the most extensively studied for their role in host- tested here, showed the peak of PG activity after  
pathogen interactions and disease development. the day of maximum growth, as it was previously  
Pectinases in their multiple forms have proved to observed in isolates of another phytopathogenic  
be important for the infection process, since they ascomycete Macrophomina phaseolina, grown in  
are the first polysaccharidases to be induced when similar culture and medium conditions (Ramos et  
fungi are grown on isolated plant cell walls, and al., 2016). PG titres obtained by C. graminicola  
are the first produced in infected tissue (Kikot et cultures in this work, resemble those attained by C.  
al., 2009; Armesto et al., 2019). The disruption lindemuthianum (0.24 U/ml) (Hugouvieux et al.,  
of pectinase genes reduces the virulence of fungi 1997), and Fusarium oxysporum f. sp. niveum (0.4  
such as Botrytis cinerea, Nectria haematococca, U/ml), while C. truncatum and F. graminearum  
Penicillium digitatum and Aspergillus niger, thus showed higher levels of PG (1.08 U/ml and 1.53 U/  
hampering their pathogenicity (Have et al., 1998; ml) (Kikot et al., 2010; Ramos et al., 2010).  
Rogers et al., 2000; López-Pérez et al., 2015; Liu  
All isolates showed β-glucosidase activity in  
et al., 2017). In this study, seven out of the eight both GAL and PEC media (0.01-0.045 U/ml)  
isolates showed PMG activity in GAL medium, (Fig. 2C). As in other phytopathogenic fungi, such  
while only four isolates presented PMG activity as Fusarium spp. (García-Maceira et al., 2000),  
in PEC medium (Fig. 2A). These isolates revealed cellulases secretion in C. graminicola occurred  
PMG activities earlier in PEC than in GAL medium. after the secretion of pectinases. The maximum  
In addition, isolates CG20 and CG22(2) showed PG of activity was detected after 15 days in all the  
activities formerly in PEC than in GAL medium. isolates, when reducing sugars levels were at their  
These findings might be attributed to catabolite lowest.  
190  
M. del P. Núñez et al. - Colletotrichum graminicola: producción de enzimas degradadoras de pared celular vegetal  
Fig. 2. Enzymatic production by C. graminicola isolates. A: Polymethylgalacturonase. B: Polygalacturonase.  
C: β-glucosidase. D: laccase, in PEC and GAL media. Numbers on top of the bars indicate the day of  
maximal activity, the values are the mean of three replications, error bars denote SEM.  
A polyphenol oxidase characterized as laccase, obtained in GAL medium, mostly before the day of  
was identified in the extracellular conidial mucilage maximum growth. Similar laccase production was  
of C. graminicola (Anderson & Nicholson, 1996) detected when growing C. truncatum in a liquid  
and might have a role in fungal survival and medium with pectin as carbon source, six out of ten  
detoxification of plant phenols. Nevertheless, the isolates produced up to 0.044 U/ml laccase (Levin  
roles of laccases in plant-pathogen interactions et al., 2007).  
remain poorly understood (Levin et al., 2007),  
The results of this study show that C.  
and mycelium associated laccase activity detected graminicola has a high intraspecific variability  
in this work might play a different function. Only when evaluating CWDEs in vitro production  
three isolates showed laccase activity in both GAL (F-values were respectively of 35.10, 10.64, 25.30  
and PEC media: CG20, CG21 and CG22(2) (Fig. and 14.02 for PMG, PG, β-glucosidase and laccase,  
2
D). The highest enzyme titres (0.063 U/ml) were with p-values <0.0001), which is consistent with  
191  
Bol. Soc. Argent. Bot. 58 (2) 2023  
the high variability observed on a genetic level bIblIogRaPhy  
when assessing ISSR markers in these same isolates  
(
Gatica et al., 2014).  
The pattern of in vitro production of CWDEs likely  
ANDERSON, D. W. & R. L. NICHOLSON. 1996.  
Characterization of laccase in the conidial mucilage  
of Colletotrichum graminicola. Mycologia 88: 996-  
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ARMESTO, C., F. G. M. MAIA, F. P. MONTEIRO & M.  
S. ABREU. 2019. Exoenzymes as a pathogenicity  
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with coffee plants. Summa Phytopathol. 45: 368-373.  
https:// doi.org/10.1590/0100-5405/191071  
reflects the in vivo pattern of enzyme production,  
and their role in the pathogenic process: initial  
pectinolytic enzyme secretion being responsible  
for allowing access to other cell wall components,  
while subsequent cellulase activity contributing to  
cell lysis and tissue maceration. Similar patterns  
were described in other phytopathogenic fungi  
(
Ten Have et al., 2002; Kikot et al., 2009). Laccase BEBBER, D. P. & S. J. GURR. 2015. Crop-destroying  
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https://doi.org/10.1016/j.fgb.2014.10.012  
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phytopathogenic fungi will contribute to finding new  
specific targets to protect plants and accelerate the  
development of new agents for battling these fungi  
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Kubicek et al., 2014).  
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notable differences in CWDEs production, not  
associated with a differential growth. This suggests  
a large intraspecific variability, which might be  
https://doi.org/10.1016/j.cropro.2013.10.005  
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pathogen. The aim of forthcoming studies will be to  
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among C. graminicola isolates causing anthracnose  
in maize are related with disparities in CWDE  
production.  
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authoR contRIbutIons  
PN and IC designed and performed the laboratory  
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authors have read and approved the final version of  
the manuscript.  
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Argentina. In: Anais do 29° Congresso Nacional de  
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to LL) and Universidad de Buenos Aires (UBA)  
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UBACYT 2020020170100163 to LL) for financial GALHAUP, C., H. WAGNER & B. HINTERSTOISSER.  
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M. del P. Núñez et al. - Colletotrichum graminicola: producción de enzimas degradadoras de pared celular vegetal  
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