Culture media to detect and criteria to evaluate and report the activity of extracellular enzymes produced by phytopathogenic fungi

Ferreira, Ana Beatriz Monteiro; Fischer, Ivan Herman; Leite, Luís Garrigós; Padovani, Carlos Roberto; Bueno, César Júnior

doi:10.1590/1808-1657000592017

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SCIENTIFIC ARTICLE, PLANT PATOLOGY • Arq. Inst. Biol. 86 • 2019 • https://doi.org/10.1590/1808-1657000592017 copy

Culture media to detect and criteria to evaluate and report the activity of extracellular enzymes produced by phytopathogenic fungi

Meios de cultura para detectar e critérios para avaliar e divulgar atividade de enzimas extracelulares produzidas por fungos fitopatogênicos

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT:

Extracellular enzymes are involved in the fungal pathogenesis in plants. Currently, culture media, data analyses, and data report related to extracellular enzymes produced in vitro conditions are different and therefore, lack standardization. This work aimed to compare the culture media cited on the literature (normal) with the potato-dextrose-agar (PDA) medium combined with a specific compound to produce extracellular enzymes through three soilborne phytopathogenic fungi (F. solani f. sp. passiflorae, S. rolfsii, and R. solani AG-4 HGI), as well as to analyze and report enzyme data based on five different criteria. The assay was randomized, with three factors (culture media, isolates, and enzymes) and six repetitions. The studied enzymes were amylase (AM), carboxymethylcellulase (CMCase), lipase (LP), laccase (LC), catalase (CT), and gelatinase (GT). The normal media detected more enzymes and was more precise compared to the PDA medium plus specific compound. The criteria that calculated the area of the circular crown of AM, CMCase, LP, and LC and measured the intensity (0 = absence, up to 4 = intense) of CT and GT adopting note scale were the best to evaluate and report the results of the enzymes. We suggest the normal media culture to study enzyme production, as well as the criteria mentioned to assess and report the data related to enzyme activities.

KEYWORDS:
enzymes; soilborne phytopathogenic fungi; methodology

Fungi	Average	Extracellular enzynes
		Note scales
		Catalase		Gelatinase
Fusarium solani f. sp. passiflorae	Normal	2.0 (2.0; 2.0)¹ a A α 2.0 (2.0; 2.0) a A α		2.0 (2.0; 2.0) b A α 1.0 (1.0; 2.0) b B α
Fusarium solani f. sp. passiflorae	PDA	2.0 (2.0; 2.0)¹ a A α 2.0 (2.0; 2.0) a A α		2.0 (2.0; 2.0) b A α 1.0 (1.0; 2.0) b B α
Sclerotium rolfsii	Normal	2.0 (2.0; 2.0) a A α 2.0 (2.0; 2.0) a A α		1.0 (1.0; 1.0) c A β 1.0 (1.0; 2.0) b A β
Sclerotium rolfsii	PDA	2.0 (2.0; 2.0) a A α 2.0 (2.0; 2.0) a A α		1.0 (1.0; 1.0) c A β 1.0 (1.0; 2.0) b A β
Rhizoctonia solani AG-4 HGI	Normal	2.0 (2.0; 2.0) a A β 2.0 (2.0; 2.0) a A β		4.0 (3.0; 4.0) a A α 4.0 (4.0; 4.0) a A α
Rhizoctonia solani AG-4 HGI	PDA	2.0 (2.0; 2.0) a A β 2.0 (2.0; 2.0) a A β		4.0 (3.0; 4.0) a A α 4.0 (4.0; 4.0) a A α
Control	Normal	1.0 (1.0; 1.0) b A α 1.0 (1.0; 1.0) b A α		1.0 (1.0; 1.0) c A α 1.0 (1.0; 1.0) b A α
Control	PDA	1.0 (1.0; 1.0) b A α 1.0 (1.0; 1.0) b A α		1.0 (1.0; 1.0) c A α 1.0 (1.0; 1.0) b A α
Fungi	Average	Extracellular enzynes
		Enzyme activity area (mm²)
		Amylase	Carboxymethylcellulase	Lipase	Laccase
Fusarium solani f. sp. passiflorae	Normal	0.0 (0.0; 0.0) a A Ω 0.0 (0.0; 0.0) a A β	290.4 (230.8; 452.2) a A β 297.1 (58.1; 429.9) a A α	638.8 (533.2; 784.2) a B β 0.0 (0.0; 0.0) a B β	0.0 (0.0; 0.0) b A Ω 0.0 (0.0; 0.0) b A β
Fusarium solani f. sp. passiflorae	PDA	0.0 (0.0; 0.0) a A Ω 0.0 (0.0; 0.0) a A β	290.4 (230.8; 452.2) a A β 297.1 (58.1; 429.9) a A α	638.8 (533.2; 784.2) a B β 0.0 (0.0; 0.0) a B β	0.0 (0.0; 0.0) b A Ω 0.0 (0.0; 0.0) b A β
Sclerotium rolfsii	Normal	0.0 (0.0; 0.0) a A β 0.0 (0.0; 0.0) a A β	0.0 (0.0; 0.0) b A β 0.0 (0.0; 0.0) b A β	0.0 (0.0; 0.0) c A β 0.0 (0.0; 0.0) a A β	388.6 (0.0; 637.4) a A α 377.5 (0.0; 450.4) a A α
Sclerotium rolfsii	PDA	0.0 (0.0; 0.0) a A β 0.0 (0.0; 0.0) a A β	0.0 (0.0; 0.0) b A β 0.0 (0.0; 0.0) b A β	0.0 (0.0; 0.0) c A β 0.0 (0.0; 0.0) a A β	388.6 (0.0; 637.4) a A α 377.5 (0.0; 450.4) a A α
Rhizoctonia solani AG-4 HGI	Normal	0.0 (0.0; 0.0) a A β 0.0 (0.0; 0.0) a A β	0.0 (0.0; 0.0) b A β 0.0 (0.0; 0.0) b A β	372.0 (337.6; 472.0) b A α 0.0 (0.0; 0.0) a B β	240.1 (155.4; 364.8) a A α 417.4 (417.4; 615.4) a A α
Rhizoctonia solani AG-4 HGI	PDA	0.0 (0.0; 0.0) a A β 0.0 (0.0; 0.0) a A β	0.0 (0.0; 0.0) b A β 0.0 (0.0; 0.0) b A β	372.0 (337.6; 472.0) b A α 0.0 (0.0; 0.0) a B β	240.1 (155.4; 364.8) a A α 417.4 (417.4; 615.4) a A α
Control	Normal	0.0 (0.0; 0.0) a A α 0.0 (0.0; 0.0) a A α	0.0 (0.0; 0.0) b A α 0.0 (0.0; 0.0) b A α	0.0 (0.0; 0.0) c A α 0.0 (0.0; 0.0) a A α	0.0 (0.0; 0.0) b A α 0.0 (0.0; 0.0) b A α
Control	PDA	0.0 (0.0; 0.0) a A α 0.0 (0.0; 0.0) a A α	0.0 (0.0; 0.0) b A α 0.0 (0.0; 0.0) b A α	0.0 (0.0; 0.0) c A α 0.0 (0.0; 0.0) a A α	0.0 (0.0; 0.0) b A α 0.0 (0.0; 0.0) b A α

Enzymes	F. solani	Control	F. solani	Control	F. solani	Control	F. solani	Control	F. solani	Control
	Criteria to evaluate extracellular enzymes
	1 - Note scale and Area (mm²)		2 - EI		3 - Pz		4 - RA		5 - Symbols and Halo (mm) + Note scale
Catalase	2.0² a B (2.0; 2.0)	1.0 a A (1.0; 1.0)	*		*		*		+	-
Gelatinase	2.0 a B (2.0; 2.0)	1.0 a A (1.0; 1.0)	*		*		*		+	-
Amylase	0.0 a A (0.0; 0.0)	0.0 a A (0.0; 0.0)	1.0 b A (1.0; 1.0)	1.0 a A (1.0; 1.0)	1.0 (N)	1.0 (N)	NSEA	NSEA	0 (0)	0 (0)
CMCase¹	290.4 b B (230.8; 452.2)	0.0 a A (0.0; 0.0)	0.92 b A (0.88; 0.94)	1.0 a A (1.0; 1.0)	0.92 (P)	1.0 (N)	NSEA	NSEA	4 (1)	0 (0)
Lipase	638.8 c B (533.2; 784.2)	0.0 a A (0.0; 0.0)	0.87 a A (0.85; 0.89)	1.0 a B (1.0; 1.0)	0.87 (P)	1.0 (N)	NSEA	NSEA	8 (2)	0 (0)
Laccase	0.0 a A (0.0; 0.0)	0.0 a A (0.0; 0.0)	1.0 b A (1.0; 1.0)	1.0 a A (1.0; 1.0)	1.00 (N)	1.0 (N)	NSEA	NSEA	0 (0)	0 (0)
Enzymes	S. rolfsii	Control	S. rolfsii	Control	S. rolfsii	Control	S. rolfsii	Control	S. rolfsii	Control
	Criteria to evaluate extracellular enzymes
	1 - Note scale and Area (mm²)		2 - EI		3 - Pz		4 - RA		5 - Symbols and Halo (mm) + Note scale
Catalase	2.0 a B (2.0; 2.0)	1.0 a A (1.0; 1.0)	*		*		*		+	-
Gelatinase	1.0 b A (1.0; 1.0)	1.0 a A (1.0; 1.0)	*		*		*		-	-
Amylase	0.0 a A (0.0; 0.0)	0.0 a A (0.0; 0.0)	1.0 a A (1.0; 1.0)	1.0 a A (1.0; 1.0)	1.0 (N)	1.0 (N)	NSEA	NSEA	0 (0)	0 (0)
CMCase¹	0.0 a A (0.0; 0.0)	0.0 a A (0.0; 0.0)	1.0 a A (1.0; 1.0)	1.0 a A (1.0; 1.0)	1.0 (N)	1.0 (N)	NSEA	NSEA	0 (0)	0 (0)
Lipase	0.0 a A (0.0; 0.0)	0.0 a A (0.0; 0.0)	1.0 a A (1.0; 1.0)	1.0 a A (1.0; 1.0)	1.0 (N)	1.0 (N)	NSEA	NSEA	0 (0)	0 (0)
Laccase	388.6 b A (0.0; 637.4)	0.0 a B (0.0; 0.0)	0.92 a A (0.89; 1.0)	1.0 a A (1.0; 1.0)	0.93 (P)	1.0 (N)	NSEA	NSEA	4 (1)	0 (0)
Enzymes	R. solani	Control	R. solani	Control	R. solani	Control	R. solani	Control	R. solani	Control
	Criteria to evaluate extracellular enzymes
	1 - Note scale and Area (mm²)		2 - EI		3 - Pz		4 - RA		5 - Symbols and Halo (mm) + Note scale
Catalase	2.0 b B (2.0; 2.0)	1.0 a A (1.0; 1.0)	*		*		*		+	-
Gelatinase	4.0 a B (3.0; 4.0)	1.0 a A (1.0; 1.0)	*		*		*		+++	-
Amylase	0.0 a A (0.0; 0.0)	0.0 a A (0.0; 0.0)	1.0 b A (1.0; 1.0)	1.0 a A (1.0; 1.0)	1.0 (N)	1.0 (N)	NSEA	NSEA	0 (0)	0 (0)
CMCase¹	0.0 a A (0.0; 0.0)	0.0 a A (0.0; 0.0)	1.0 b A (1.0; 1.0)	1.0 a A (1.0; 1.0)	1.0 (N)	1.0 (N)	NSEA	NSEA	0 (0)	0 (0)
Lipase	372.0 c A (337.6; 472.0)	0.0 a B (0.0; 0.0)	0.89 a B (0.87; 0.91)	1.0 a A (1.0; 1.0)	0.89 (P)	1.0 (N)	NSEA	NSEA	5 (1)	0 (0)
Laccase	240.1 b A (155.4; 364.8)	0.0 a B (0.0; 0.0)	0.89 a B (0.83; 0.91)	1.0 a A (1.0; 1.0)	0.87 (P)	1.0 (N)	NSEA	NSEA	5 (1)	0 (0)

Criterion 1 - Calculation of the circular crown area. Formula described by GIECK (without yearGIECK, K. Manual de fórmulas técnicas. São Paulo: Hemus.). This formula was used to quantify the enzymes amylase, carboxymethylcellulase, lipase, and laccase. The catalase and gelatinase enzymes were quantified by symbols describing their intensity, which were later transformed into a note scale, with 1 = absent (or - = symbol: absent); 2 = weak (or + = symbol: weak); 3 = moderate (or ++ = symbol: moderate); 4 = intense (or +++ = symbol: intense). For catalase, the intensity refers to the size of bubble formation and for gelatinase, the liquefaction or not of the medium;
Criterion 2 - Calculation of the enzymatic index (EI). Formula described by BOCCHESE et al. (2003BOCCHESE, C.A.C.; MARTINELLI, J.A.; MATSUMURA, A.T.S.; FEDERIZZI, L.C.; PRESTES, A.M. Virulência, atividade enzimática e padrões de isoesterases de isolados de Pyrenophora chaetomioides, agente etiológico da mancha de grãos e folhas de aveia. Fitopatologia Brasileira, Brasília, v.28, n.1, p.11-16, 2003. http://doi.org/10.1590/S0100-41582003000100002
http://doi.org/10.1590/S0100-41582003000... ). This index was applied to the enzymes amylase, carboxymethylcellulase, lipase, and laccase;
Criterion 3 - Calculation of the Pz index. Formula described by HANKIN; ANAGNOSTAKIS (1975HANKIN L.; ANAGNOSTAKIS, S.L. The use of solid media for detection of enzyme production by fungi. Mycologia, New York, v.61, n.3, p.597-607, 1975. https://doi.org/10.2307/3758395
https://doi.org/10.2307/3758395... ). According to the index, enzyme activity is negative (N) when Pz = 1, positive (P) when 0.64 ≤ Pz < 1, and strongly positive (SP) when Pz < 0,64. This index was applied to the enzymes amylase, carboxymethylcellulase, lipase, and laccase;
Criterion 4 - Calculation of the RA index. Formula and criterion described by KRISHNAN et al. (2011KRISHNAN, A.; ALIAS, S.A.; WONG, C.M.V.L.; KA-LAI, P.; CONVEY, P. Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica. Polar Biology, Berlin, v.34, p.1535-1542, 2011. https://doi.org/10.1007/s00300-011-1012-3
https://doi.org/10.1007/s00300-011-1012-... ) and other authors. If RA > 1, enzymatic activity (SEA) is significant, and if RA < 1, enzymatic activity is not significant (NSEA). This index was applied to the enzymes amylase, carboxymethylcellulase, lipase, and laccase;
Criterion 5 - Method described by BASTOS (2005BASTOS C.N. Produção de enzimas extracelulares por Crinipellis perniciosa. Fitopatologia Brasileira, Brasília, v.30, n.3, p.286-288, 2005. http://doi.org/10.1590/S0100-41582005000300011
http://doi.org/10.1590/S0100-41582005000... ). Measurement of the diameter of the halo formed around the colonies, represented by the note scale: (0), no enzyme production; (1), halo with diameter between 1-5 mm; (2), diameter between 5-10 mm; (3), diameter between 10-20 mm; (4), diameter between 20-30 mm; e (5), diameter higher than 30 mm. This method was applied to the enzymes amylase, carboxymethylcellulase, lipase, and laccase. To evaluate the enzymes catalase and gelatinase, BASTOS (2005BASTOS C.N. Produção de enzimas extracelulares por Crinipellis perniciosa. Fitopatologia Brasileira, Brasília, v.30, n.3, p.286-288, 2005. http://doi.org/10.1590/S0100-41582005000300011
http://doi.org/10.1590/S0100-41582005000... ) used symbols for a subjective estimation of enzyme production, based on the intensity of the enzymes formed in the medium: +++ intense; ++ moderate; + weak; and - absent;
^*The method does not contemplate enzyme evaluation; ¹carboxymethylcellulase; ²median and maximum and minimum values of the interquartile half-amplitude data.
Different lowercase letters indicate significance (p < 0.05) among enzymes for each fungus; different uppercase letters indicate significance of the fungus (p < 0.05) within the enzyme, according to Dunn’s multiple comparison test with the Bonferroni correction (ZAR, 2009ZAR, J.H. Biostatistical analysis. 5. ed. New Jersey: Prentice-Hall. 2009. 994p.).

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[1] *Corresponding author: cjbueno@biologico.sp.gov.br