Enzymatic activity of fungi isolated from crops.

AIM: To detect and assess the activity of extracellular hydrolytic enzymes and to find differences in enzymograms between fungi isolated from wheat and rye samples and grown on Czapek-Dox Broth and Sabouraud Dextrose Broth enriched with cereal (wheat or rye). Isolated strains were also classified in the scale of biosafety levels (BSL).
MATERIAL AND METHODS: The study used 23 strains of fungi cultured from samples of wheat and rye (grain, grain dust obtained during threshing and soil) collected in the Lublin region (eastern Poland). API ZYM test (bioMérieux) was carried out according to the manufacturer's instructions. Classification of BSL (Biosafety levels) was based on the current literature.
RESULTS: High enzymatic activity was found in strains cultured in media containing 1% of wheat grain (Bipolaris holmi, Penicillium decumbens) and with an addition of 1% of rye grain (Cladosporium herbarum, Aspergillus versicolor, Alternaria alternata). The total number of enzymes varied depending on the type of media, and in most cases it was higher in the culture where an addition of cereal grains was used.
CONCLUSIONS: Isolated strains of fungi reveal differences in the profiles of the enzyme assay. It can be assumed that the substrate enriched in grains stimulate the higher activity of mold enzymes.

Introduction

API ZYM test (bioMérieux) is a semiquantitative assay allowing to assess the presence and activity of 19 hydrolytic enzymes. The release of hydrolytic enzymes into the environment by dermatophytes, yeasts, and molds is an important component in the pathogenesis of infection. Hydrolases (esterases, sulfatases, glycosidases and peptidases) facilitate the degradation of keratin, and thus the penetration of the mycelium into tissues [1-3].

The isolated and identified strains were also assigned to specific classes of biosafety. The Classification of Biosafety Level (BSL) determines the safety scale of potentially pathogenic fungi for humans and animals by highlighting four hazard classes. The class BSL-1 represents saprophytes or plant pathogens causing a coincidental, superficial, non-invasive or benign threat [4]. However, this may cause non-infectious respiratory diseases of an allergic or immunotoxic nature [5]. Class BSL-2 species are characterized by a relatively high ability to survive in the tissues of vertebrates, and in patients with severe immune disorders can cause a deep, opportunistic infection. Pathogens belonging to the class of BSL-3 are potentially capable of causing severe and deep fungal infections in apparently healthy subjects [4].

Aim

The aim of the study was to detect and assess the activity of extracellular hydrolytic enzymes and to find differences in enzymograms between fungi isolated from wheat and rye samples and grown on Czapek-Dox Broth and Sabouraud Dextrose Broth enriched with cereal (wheat or rye). Isolated strains were also classified in the scale of biosafety levels (BSL).

Material and methods

The study used 23 strains of fungi isolated from samples of grain, grain dust obtained during threshing and soil from the crops of wheat and rye collected in the Lublin area. In order to prepare the isolates for the API ZYM test, the strains were grown initially on two solid media: Malt Agar (Becton, Dickinson and Co.) intended for all kinds of fungi and Potato Dextrose Agar (Becton, Dickinson and Co.) which is a selective medium for Fusarium spp. Samples were incubated at 24°C or 30°C for 72 h depending on the medium, and then at room temperature until all have been producing spores. Then the biopsy was taken (5 mm in diameter) from the margin of the culture and transferred to four liquid media: Czapek-Dox Broth (Becton, Dickinson and Co.) pure and supplemented with 1% of grain (wheat or rye), and Sabouraud Dextrose Broth (Becton, Dickinson and Co.) pure and supplemented with 1% of grain. Media without the tested isolates were used as controls. The isolates were then incubated for 24 days at 24°C. Supernatants obtained from culture (after centrifugation or sedimentation) were tested. Supernatants from control cultures were used as a control. API ZYM tests were performed according to the manufacturer’s instructions by placing 65 µl of the supernatant at appropriate points of the test strip. Reading was made visually. Enzyme activity was determined using a scale of 0 to 5, with 0 indicating a negative reaction. Differences in enzyme activity were compared in cultures with and without addition of the grain extract and statistically tested with Mann-Whitney-Wilcoxon test (R statistical software environment version 3.0.2) [6].

With the help of available literature the tested isolates were classified into classes of biosafety (BSL) [7].

Results

API ZYM test performed on all the control substrates tested showed no activity of hydrolytic enzymes. The following strains isolated from cultures were characterized by the greatest number of active hydrolytic enzymes: Exserohilum sp., Bipolaris holmi (Tables 1 and 2) in strains isolated from samples of wheat and Aspergillus versicolor, Penicillium chrysogenum, Alternaria alternata isolated from samples of rye (Tables 3 and 4). The highest activity of hydrolytic enzymes (4 or 5 in the adopted scale of activity) was found in the strains of Bipolaris holmi and Penicillium decumbens which were grown on media containing 1% of wheat grains (Tables 1 and 2); and in strains of Cladosporium herbarum, Aspergillus versicolor, and Alternaria alternata grown on media containing 1% of rye grains (Tables 3 and 4). In isolates from wheat samples the most active enzymes were N-acetyl-β-glucosyloamidase, β-glucosidase, acid phosphatase (Table 1) while in isolates from rye samples, β-glucosidase, N-acetyl-β-glucosyloamidase, and alkaline phosphatase exhibited the highest activity (Table 3).

Table 1

Activity of mold exoenzyme isolated from wheat on media with and without addition of wheat

Isolate	Medium	Enzyme activity*,**
		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19
Alternaria alternata	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	0405	2204	3304	0000	2514	1513	0101	4210	0201	1405	1515	2020	0003	0203	1314	5555	5525	0001	0001
Aspergillus fumigatus	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	1031	2121	3012	1000	1353	1412	1022	1012	1022	5355	4442	5513	3333	0000	2403	3533	5344	1043	0000
Aspergillus glaucus	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	3253	2232	3242	1000	0041	0132	0030	0030	0031	5252	5253	5453	2145	0040	1003	5245	4345	1242	0000
Aspergillus sydowii	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	2525	1211	2232	1010	0513	0222	0222	3231	2220	3545	5553	5512	1010	0002	1000	3323	3333	3313	2010
Aspergillus tamarii	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	0123	2222	1120	1000	0123	1002	1000	1101	0100	2212	1222	0022	0013	0013	1223	3434	4433	0000	0000
Aspergillus terreus	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	0314	1211	0000	0000	0424	0122	0011	0001	0000	3414	3413	2524	0100	1303	0201	2443	2344	0423	0000
Aspergillus versicolor	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	3132	0011	0010	0000	0000	0000	0000	0000	0000	4242	1115	1241	0101	0000	0020	5442	2040	0030	0000
Bipolaris holmi	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	1345	0233	2232	0212	1545	0222	0230	0353	0010	2253	2353	4202	3245	0205	3332	5555	5555	1212	1202
Exserohilum sp.	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	4133	3122	3012	1100	5242	5120	5020	1432	1122	4053	5253	2453	1432	1032	3255	5553	4435	1132	0020
Fusarium proliferatum	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	4343	1111	1223	0100	2545	0011	0110	1000	0101	2433	2334	2503	1402	0000	0100	3533	3523	1300	0000
Fusarium tricinctum	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	1445	1223	2234	0010	0453	0020	0000	0200	0000	3535	1235	2215	0200	0000	0001	2515	3445	0102	0000
Penicillium decumbens	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	0103	2202	1202	0002	3213	0002	0000	0000	0000	5445	4325	4405	5555	5515	4405	5555	4454	1204	0000
Penicillium expansum	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	0001	1102	0203	0001	2204	0002	1110	0000	0000	2315	3325	2305	4504	1405	1304	3414	3434	0002	0000
Scopulariopsis brevicaulis	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	0101	2234	5434	2302	3413	5534	2101	0000	0000	0100	1111	0001	1100	0000	0001	1515	0415	0233	0000
Scopulariopsis brumptii	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	0102	3303	3404	1201	1505	3503	0000	0000	0000	0020	1112	0000	1405	0000	0002	1515	1424	1102	0000
Stemphylium sp.	Czapek-DoxCzapek-Dox + wheatSabouraudSabouraud + wheat	0154	2022	4111	1000	2123	1022	0100	1112	0001	1353	1132	1010	0103	0011	1203	5555	5545	0000	0000

Enzymes: 1) alkaline phosphatase, 2) esterase (C4), 3) esterase lipase (C8), 4) lipase (C14), 5) leucine arylamidase, 6) valine arylamidase, 7) cystine arylamidase, 8) trypsin, 9) α-chymotrypsin, 10) acid phosphatase, 11) naphthol-as-bi-phosphohydrolase, 12) α-galactosidase, 13) β-galactosidase, 14) β-glucuronidase, 15) α-glucosidase, 16) β-glucosidase, 17) N-acetyl-β-glucosaminidase, 18) α-mannosidase, 19) α-fucosidase.

0 – no activity, 1–5 – activity increases with color intensity (1 low activity, 5 high activity).

Table 2

Enzyme activity in isolates from wheat crops

Isolate	Total enzyme activity summed up				Number of active enzymes
	Czapek-Dox	Czapek-Dox + wheat	Sabouraud	Sabouraud + wheat	Czapek-Dox	Czapek-Dox + wheat	Sabouraud	Sabouraud + wheat
Alternaria alternata	28	49**	23	50***	12	13	13	14
Aspergillus fumigatus	40	35	41	41	17	10	15	16
Aspergillus glaucus	37*	23	63*^^	39^	12	11	16	14
Aspergillus sydowii	37	46	35	37	15	14	17	14
Aspergillus tamarii	18	21	23	33^	11	11	12	13
Aspergillus terreus	14	40**	21	38*	7	13	11	14
Aspergillus versicolor	16	11	27^	14	6	6	10	7
Bipolaris holmi	30	49*	54^	56	12	18	16	17
Exserohilum sp.	54	33	58*	41	18	14	18	15
Fusarium proliferatum	23	44**^	24	32	12	15	10	12
Fusarium tricinctum	15	35**	29^	43	8	12	11	11
Penicillium decumbens	43^	43	23	57**^^	12	13	7	15
Penicillium expansum	23^^	35*	8	51***^^	11	12	5	15
Scopulariopsis brevicaulis	22	34	16	35**	9	13	8	13
Scopulariopsis brumptii	16	35*	6	38**	10	11	4	12
Stemphylium sp.	25	22	32	37^^	12	11	12	14
Mean	27.6	34.7	30.2	40.1	11.5	12.3	11.6	13.5

Mann-Whitney-Wilcoxon test for the difference of enzyme activity was performed. Asterisks (*) are stated at the higher number to show the significant difference between pure and supplemented medium and carets (^) are stated at the higher number to show the difference between Czapek-Dox and Sabouraud media (pure and supplemented). The significance of differences:

(^) p < 0.05;

(^^) p < 0.01;

(^^^) p < 0.001.

Table 3

Activity of mold exoenzyme isolated from rye on media with and without addition of rye

Isolate	Medium	Enzyme activity[*,**]
		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19
Alternaria alternata	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	0524	2323	4413	0002	2525	2313	0100	3402	0000	1313	1323	1423	1015	0000	2424	4535	5535	0010	0000
Aspergillus versicolor	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	4445	1324	2424	0001	1414	1525	0111	0000	0101	4514	2422	5515	3515	0000	0114	3555	3524	0124	0000
Cladosporium herbarum	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	2505	2213	1404	0000	0503	0403	0001	0001	0001	1505	1405	3515	0000	0405	0201	4515	4534	0405	0305
Fusarium cerealis	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	1524	2312	2201	0000	1403	0302	0000	4100	0100	1515	1423	0301	1200	0000	0102	5534	0044	0103	0000
Fusarium oxysporum	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	0334	1312	2213	1010	3414	0403	0100	1414	0100	0535	0523	0100	1304	0000	0001	2555	2050	0201	0000
Fusarium tricinctum	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	2435	2103	2112	0000	1313	0201	0000	1100	0000	4425	0445	0013	0202	0000	0002	4334	0035	0002	0000
Penicillium chrysogenum	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	0504	1402	1203	0000	5505	2403	0100	0000	0503	0505	1525	0505	2424	0000	3515	4535	4414	0304	2304
Penicillium diverse	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	4524	0302	0401	0000	1535	4401	0100	0000	0100	3515	2525	0405	0303	0200	0304	5335	4434	0405	0000
Rhizopus oryzae	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	2545	4233	3333	0000	0321	0330	0020	0000	0000	0531	1532	1425	1503	0000	0000	1545	2105	1223	0000
Ulocladium chartarum	Czapek-DoxCzapek-Dox + ryeSabouraudSabouraud + rye	1235	3234	4210	0000	0345	0334	0101	0100	0000	2124	3113	0233	0415	0313	1534	4555	4555	0001	0000

Enzymes: 1) alkaline phosphatase, 2) esterase (C4), 3) esterase lipase (C8), 4) lipase (C14), 5) leucine arylamidase, 6) valine arylamidase, 7) cystine arylamidase, 8) trypsin, 9) α-chymotrypsin, 10) acid phosphatase, 11) naphthol-as-bi-phosphohydrolase, 12) α-galactosidase, 13) β-galactosidase, 14) β-glucuronidase, 15) α-glucosidase, 16) β-glucosidase, 17) N-acetyl-β-glucosaminidase, 18) α-mannosidase, 19) α-fucosidase;

0 – no activity, 1–5 – activity increases with color intensity (1 low activity, 5 high activity).

Table 4

Enzyme activity in isolates from rye crops

Isolate	Total enzyme activity summed up				Number of active enzymes
	Czapek-Dox	Czapek-Dox + rye	Sabouraud	Sabouraud + rye	Czapek-Dox	Czapek-Dox + rye	Sabouraud	Sabouraud + rye
Alternaria alternata	27^	48*	14	54**	11	14	8	16
Aspergillus versicolor	29	53**	27	58**	11	15	14	16
Cladosporium herbarum	18^	57**	6	61***	8	14	4	17
Fusarium cerealis	18	40*	13	34**	9	14	6	12
Fusarium oxysporum	13	43**	23	39*	8	14	10	12
Fusarium tricinctum	16	25	18	42**^	7	10	8	13
Penicillium chrysogenum	25^	65**	9	61***	10	16	5	15
Penicillium diverse	23	56**	14	49**	7	16	6	13
Rhizopus oryzae	16	43*	31^	36	9	12	11	11
Ulocladium chartarum	22	40	35	52**	8	15	13	14
Mean	20.7	47.0	19.0	48.6	8.8	14.0	8.5	13.9

Mann-Whitney-Wilcoxon test for the difference of enzyme activity was performed. Asterisks (*) are stated at the higher number to show the significant difference between pure and supplemented medium and carets (^) are stated at the higher number to show the difference between Czapek-Dox and Sabouraud media (pure and supplemented). The significance of differences:

(^) p < 0.05;

(^^) p < 0.01;

(^^^) p < 0.001.

Comparing the activity of extracellular enzymes in the medium with and without the addition of grain, it was found that supplementation with cereal grain (wheat, rye) resulted in increased activity of the enzymes produced by the isolates in most cases. From 16 isolates of wheat crops in Czapek-Dox medium with an addition of wheat, in 7 cases statistically significant higher enzyme activity was found, and in one case statistically significant lower enzyme activity was found as compared to pure Czapek-Dox medium. In the Sabouraud medium with an addition of wheat in 6 cases statistically significant higher activity was found, and in 2 cases statistically significant lower activity was detected comparing to pure Sabouraud medium (Tables 1 and 3). The differences in isolates from rye were more distinct than in Czapek-Dox medium with an addition of rye: 8 from 10 isolates showed statistically significant higher activity, and in Sabouraud medium significant higher activity was found in 9 isolates (Tables 3 and 4). When differences between media were compared the differences were much smaller. In wheat isolates higher activity in pure media was found in 4 Sabouraud cultures and in two Czapek-Dox cultures, and in supplemented media 5 Sabouraud cultures showed higher enzyme activity and one Czapek-Dox culture. The differences between media in rye isolates were smaller.

The vast amount of isolates was assigned to BSL-1 (Table 5), i.e. to the class which represents saprophytes or plant pathogens causing a non-invasive or mild threat.

Table 5

Biosafety levels of isolated molds [7]

No.	Isolate	BSL
1	Alternaria alternata	I
2	Aspergillus fumigatus	II
3	Aspergillus glaucus	I
4	Aspergillus sydowii	I
5	Aspergillus tamarii	I
6	Aspergillus terreus	II
7	Aspergillus versicolor	I
8	Bipolaris holmi	ND
9	Cladosporium herbarum	I
10	Exserohilum sp.	ND
11	Fusarium cerealis	ND
12	Fusarium oxysporum	II
13	Fusarium proliferatum	I
14	Fusarium tricinctum	ND
15	Penicillium chrysogenum	I
16	Penicillium decumbens	I
17	Penicillium diverse	ND
18	Penicillium expansum	I
19	Rhizopus oryzae	I
20	Scopulariopsis brevicaulis	II
21	Scopulariopsis brumptii	II
22	Stemphylium sp.	ND
23	Ulocladium chartarum	I

ND – not defined.

Discussion

One of molds, which had high enzymatic activity, belonged to the genus Exserohilum, which consists of more than 30 species. These saprophytes are commonly found on the remains of plants, some of which can cause diseases of plants, animals and humans (immunocompromised persons are particularly at risk) [8]. Another mold showing high activity of the enzymes was species from Bipolaris genus. This type includes more than 100 species, most of them are saprobiotic species found in the soil and some of them have the potential pathogenicity to animals and humans [9]. A significant amount of hydrolytic enzymes was also found in an isolate from the genus Alternaria, the causative agent of phaeohyphomycosis or onychomycosis, sinusitis, ulcerative skin infections, corneal inflammation, and deep mycosis. Infections caused by this genus are a growing problem in patients who have been treated with immunosuppressants. It is also a common allergen [10]. Aspergillus versicolor is described as a rare cause of various mycoses in humans. It is often isolated from fungal diseases of domestic animals and habitats of various soil [3]. The genus Cladosporium is very rarely a cause of opportunistic infections in humans. It is a common allergen [10]. Cladosporium herbarum is the most common species in this genus, inhabiting dead organic remains of plants in soil habitats. Molds from the genus Penicillium occur commonly in the fields and meadows, and is also found in flats and basements. It is often present in fruit juices, on citrus and moldy bread. This can cause infections particularly in people with immunodeficiency. High concentration of spores of this fungus in the air can act as an allergen. P. chrysogenum species has a worldwide range. It was isolated many times from soil in different climatic zones, including the polar zone [3].

Hydrolytic enzymes that are released into the environment by dermatophytes, yeasts, and molds are an important element in the pathogenesis of infection [11]. The enzymatic activity of fungi can colonize most surfaces dead or living. The enzymes produced by fungi allow the degradation and the use of most of the naturally occurring compounds and synthetic substances. Decomposition processes are initiated by adaptive enzymes, produced in response to the “signal” from the environment. Changing the proportions of trophic components in the substrate or appearance of trace amounts of a new substrate, stimulates production of specific enzymes [12]. The hydrolases which are synthesized by pathogenic fungi include esterases, and they include the carboxyl group ester hydrolases (triacylglycerol lipase, phospholipase A2), the phosphoric monoester hydrolases (alkaline phosphatase, acid phosphatase) and sulfuric ester hydrolases (sulfatases); glycosidases (α-glucosidase, β-glucosidase, α-mannosidase); peptidases – exopeptidases (proteinases, e.g. aspartic proteinase, aminopeptidases, e.g. arylamidase) and endopeptidase (proteinase hydrolyzing C-N bond, e.g. urease). Using a commercial standardized test (API ZYM, bioMérieux), one can create a different fungal species enzymogram illustrating the characteristic enzymatic activity [3, 13–15].

Obtained results tend to reflect methods of storage and processing of grain; whether the technology completely eliminates molds from food. As the results show, some molds (e.g. Exserohilum sp., Bipolaris holmi, Alternaria alternata, Cladosporium herbarum, Aspergillus versicolor, Penicillium chrysogenum) isolated from samples of grain, grain dust, and soil from crops of wheat and rye, probably will also be able to show significant enzyme activity in natural environment, which may help to adapt to new conditions, and to colonize various locations and affect human health (especially in immunocompromised persons) exposed to inhalation of large amounts of dust generated during harvesting grain cereals (i.e. threshing with a combine harvester or pouring grain into barns).

Conclusions

Hydrolytic exoenzyme activity in tested fungi depends on the type of the medium and the addition of the grain extract. Particular grains can stimulate activity of extracellular enzymes in tested fungi. Activation of enzymes was influenced by an addition of wheat and rye to the medium. The relationship between high enzymatic activity of examined fungi and BSL scale was not found.