2.1. Endogenous Mycobiota of Barley
The filamentous fungi identified from surface disinfected barley grain before and during storage in silo are listed in
Table 1, whereas the isolation frequency and relative density are presented in
Table 2. The results indicate that mycological colonization of barley grain before and during storage may be diverse and a subject to change during storage. From the barley samples taken immediately after harvest, there were isolated 8 genera and
Mycelia sterilia (isolates without sporulation) of specific microscopic fungi belonging to the field ecosystem. Of all the 142 strains found, species of
Alternaria (58% RD) and
Fusarium (13% RD) predominated, particularly in the Laudis variety. Higher genera representation (7) was obtained from the Kangoo variety, but the highest number of isolates was from the Laudis variety. Isolates of genera
Alternaria, Arthrinium, and
Epicoccum were found in all tested samples.
Compared to the baseline of freshly harvested grain, a small increase of detected filamentous fungi was observed in barley stored for three or six months, except for the Laudis variety with the number of isolates decreased at that length of storage. A total of 146 strains from 10 genera and Mycelia sterilia were identified in barley stored for 3 months. There were negligible differences in the genera representation of filamentous fungi, but the highest number of isolates was found in the Laudis variety. Alternaria was the most frequently observed/isolated genus with 45 isolates and RD of 31% of all the isolates found. Cladosporium and Rhizopus were the second predominant genera with RD of 19%, each.
After 6 months of barley grain storage, the number of microfungi increased slightly to a total of 149 isolates with a comparable representation of 10 genera and Mycelia sterilia to those detected after 3 months of storage. In all samples, the genera Alternaria, Arthrinium, Epicoccum, Fusarium, Penicillium, and Rhizopus were detected. The RD of genus Alternaria was the highest (44%). From five distinct Penicillium species detected, the most frequent and the most abundant was P. griseofulvum. The numbers of genera of filamentous microscopic fungi detected in each barley variety reached their maximum at this storage period: 9 genera and Mycelia sterilia for the Wintmalt variety and 8 genera for the Laudis and Kangoo varieties, respectively.
The barley grain samples were contaminated comparably less with the filamentous microfungi at 9 months of storage (133 isolates). On the other hand, the most fungal genera (11) were detected in these samples. The highest RD was observed for the genus Alternaria (40%), followed by Cladosporium (19%), and Penicillium (10%). The highest amount of Alternaria isolates (24) was again isolated from the Laudis variety. Genera Alternaria, Arthrinium, Cladosporium, Epicoccum, and Penicillium were detected in all samples at 9 months of storage.
The data in
Table 2 also show that, from the barley grains stored in silo for a period of 3 to 9 months, 13 fungal genera and
Mycelia sterilia were identified (428 isolates).
Alternaria,
Arthrinium, and
Penicillium were the main components of the mycobiota from 3 samples (100% IF), followed by
Epicoccum (89%),
Cladosporium,
Fusarium, and
Rhizopus (78%, each).
Alternaria was the most abundantly detected genus (163 isolates) with RD of 38%. The isolated
Penicillium consisted of eight species.
Penicillium brevicompactum and
P. griseofulvum showed the highest IF (33%) and RD (13%) of all the isolates from this genus.
Analysis of the detected filamentous fungi contaminants of barley showed that the Laudis barley variety had slightly higher number of isolates (47) and of genera (9) in comparison with Kangoo or Wintmalt varieties (
Table 3).
Alternaria,
Arthrinium,
Cladosporium,
Fusarium, and
Penicillium were detected in all samples. The most abundant genus was
Alternaria (39%), followed by
Fusarium (15%), and
Arthrinium (12%). During the survey, 7 isolates of 4
Penicillium species (
P. crustosum,
P. glabrum,
P. griseofulvum, and
P. chrysogenum) were isolated and identified.
In the samples stored in floor warehouse, comparatively fewer filamentous fungi contaminants were detected than in the samples stored in grain storage silo. A total of 134 strains from 10 genera and
Mycelia sterilia were identified at 3 months of storage (
Table 4).
A total of 89 isolates from 8 genera and
Mycelia sterilia were isolated from samples stored for 6 months.
Alternaria and
Penicillium were detected in all samples. Alternaria was the most abundant genus (55%), especially in the Laudis variety. Compared with samples stored 6 months, a decrease of filamentous fungi detected was recorded at 9 months of storage in both types of storage. A total of 84 strains from 7 genera were identified from barley stored in floor warehouse for 9 months.
Alternaria and
Arthrinium were detected in all the samples examined. The occurrence of
Penicillium spp. was generally low, but rich on species:
P. griseofulvum,
P. hordei,
P. chrysogenum, and
P. raistrickii. The lowest qualitative (4 genera) and quantitative (19 isolates) representation of microfungi was isolated from the Kangoo variety (
Table 3). The most abundant genera were
Alternaria and
Arthrinium (42%, each).
A total of 307 strains from 10 genera and Mycelia sterilia were identified from the barley grain samples stored in floor warehouse over a period of 3 to 9 months. The most frequent were Alternaria (100% IF), Penicillium (89%), Arthrinium (78%), and Epicoccum (67%). The most abundant genera found by descending order were Alternaria (44% RD), Arthrinium (17%), and Fusarium (7%). The isolated Penicillium strains included/encompassed 7 species, of which Penicillium griseofulvum achieved the highest IF (33%) and RD (23%).
2.2. Endogenous Mycobiota of Malt
Samples of barley grains fresh from the harvest and before storage, and of barley grains stored in silo for 3, 6, and 9 months were malted and these samples were examined for endogenous mycobiota by plating method with surface disinfection. The filamentous fungi identified in malt are indicated in
Table 5, and their IF and RD are presented in
Table 6. The total count of microscopic fungi isolated from grains malted directly after harvest was the highest (112) with 7 genera and
Mycelia sterilia, compared to malts prepared from barley stored in silo.
Alternaria, Epicoccum, and
Fusarium were the dominant fungal genera detected in the control malt.
Alternaria was the most abundant occurring genusthere, with RD of 45%, but their presence in the Laudis and Kangoo malt were lower compared to the non-malted barley grain samples. The second abundant genus was
Epicoccum (17%), followed by
Fusarium (16%). Interestingly, the Wintmalt variety had the highest number of isolates. We isolated toxicogenic
Penicillium griseofulvum and
P. chrysogenum from the Wintmalt variety only.
The endogenous mycobiome in malts prepared from barley stored for 3 months included 63 isolates from 8 genera and Mycelia sterilia. Alternaria was the most abundant occurring genus with 22 isolates and RD 35% of all the isolates found, followed by Mucor (32% RD). Their IF was the highest in the Wintmalt variety.
Isolation of fungi from malts from surface-sterilized barley grains stored for 6 months resulted in collecting of 94 fungal isolates from 8 genera and Mycelia sterilia. Alternaria, Cladosporium, Epicoccum, and Mucor were the predominant genera in all 3 samples of malt. Alternaria scored the highest RD (65%) and was the main component of Wintmalt and Laudis malt mycobiota.
The overall mycological colonization of 3 tested malts from barley stored for 9 months was the lowest. Within 54 isolates of filamentous fungi, the most abundant were Alternaria (35% RD) and Rhizopus (28% RD). Alternaria, Cladosporium, Epicoccum, Mucor, and Rhizopus were more frequently isolated from malts (67% IF), and the remaining 2 genera and Mycelia sterilia were found only in one sample of malt.
A total of 211 isolates from 10 genera and Mycelia sterilia were identified from malts prepared from barley stored in grain storage silo for 3 to 9 months. The most frequent were Alternaria (89% IF), Mucor (78% IF), Cladosporium, and Epicoccum (67% IF, each). The most abundant genera found were Alternaria (48% RD), Mucor (14%), and Rhizopus (8%).
All the previous fungal genera except
Absidia were also isolated from malted barley grain samples stored in floor warehouse (
Table 7). Nevertheless, their relative abundances varied (
Table 8). Isolates from malted barley stored in floor warehouse for 3 months included 93 strains from 7 genera of filamentous fungi and
Mycelia sterilia with 100% isolation frequency for
Alternaria and
Mucor.
Alternaria isolates predominated (55% RD), followed by
Cladosporium (12% RD),
Mucor, and
Penicillium (10% RD, each). The incidence of
Penicillium isolates revealed the occurrence of 4 different
Penicillium species with maximum relative density for
P. corylophilum (44% RD) of all the
Penicillium isolates. Samples of malt prepared from barley grains stored for 6 months in floor warehouse had the lowest number (26) and genera (3) of filamentous fungi. The highest RD was again reached by the genus
Alternaria (81%) which was present in all 3 samples. Microfungi in malt samples from barley grains stored in floor warehouse for 9 months included 73 isolates from 8 genera. The most common fungal genus was
Epicoccum (100% IF) and the highest RD was reached by the genera
Rhizopus (29%), followed by
Alternaria and
Penicillium (19% RD, each). Overall, 192 isolates from 10 genera and
Mycelia sterillia were identified in malts prepared from barley stored in floor warehouse over a period of 3 to 9 months, very similar to the malt samples from grain stored in silo. The most frequent were
Alternaria (78% IF),
Epicoccum, Mucor (56% IF, each),
Cladosporium, and
Penicillium (44% IF, each). The most abundant genera found were
Alternaria (45% RD),
Penicillium, and
Rhizopus (12% RD, each). From the 23
Penicillium isolates identified, the most frequent was
P. corylophilum (22% IF) with the highest RD being 17% of all the
Penicillium isolates.
Thirteen different genera and
Mycelia sterilia were isolated from 3 varieties of freshly harvested barley grains before storage (
Table 9). The total filamentous fungal count on control grains before storage ranged from 3.6 × 10
2 to 3.6 × 10
3 CFU/g and the total yeast count from 1.3 × 10
3 to 9.1 × 10
4 CFU/g.
Gradually, at the 6th month of storage, the grains mycobiota increased in incidence of the Penicillium species, due to storage conditions such as high atmospheric humidity. The barley grains associated mycobiota consisted of various species of fungi such as Alternaria, Arthrinium, Aspergillus, Cladosporium, Fusarium, Penicillium, Ulocladium, Mycelia sterilia, Candida, Issatchenkia, Rhodotorula, Sporobolomyces, and Wickerhamomyces belonging with predominance to the Penicillium, Aspergillus, and Rhodotorula genera. Yeasts from the analyzed samples were identified using MALDI-TOF MS Biotyper. Penicillium strains were isolated from all samples. The genus Penicillium was represented by eight species: P. aurantiogriseum, P. canescens, P. citrinum, P. glabrum, P. griseofulvum, P. polonicum, and P. raistrickii. At the 6th month of storage, the yeast abundance reached its maximum in all monitored varieties. The highest increase was observed in the Laudis variety with the yeast population ranging from 1.3 × 103 CFU/g from the freshly harvested barley grain samples to 2.4 × 107 CFU/g. A slight decrease compared to this maximum count in yeasts and filamentous fungi was observed at the 9th month of storage. Cladosporium was the predominant genus, followed by Alternaria, Arthrinium, Aspergillus, Rhizopus, and Rhodotorula. Penicillium was comparably less frequently isolated. Rhodotorula isolates occurred in the barley varieties Laudis and Kangoo and Candida and Sporobolomyces in the variety Wintmalt throughout the storage period in both types of storage.
The studies of barley grains stored in floor warehouse revealed that the initial yeasts count increased at 3rd and 6th months of storage and slightly decreased at 9th months of storage. At the 6th month of storage, the yeast abundance reached maximum in all monitored varieties, and in the 9th month decreased. The yeast population isolated from barley grains ranged from 1.3 × 103 CFU/g from freshly harvested samples to 1.1 × 106 CFU/g in the Wintmalt variety stored for 6 months. Samples were less contaminated with fungi. At the 3rd month of storage, the microfungi abundance reached maximum and included Alternaria, Arthrinium, Aspergillus, Cladosporium, Epicoccum, Fusarium, Penicillium, and Mycelia sterilia. In the Kangoo variety stored for 3 months, fewer filamentous fungi were detected than from the freshly harvested samples. Saprophytic fungi such as Arthrinium and Aspergillus, isolated from samples at 3 months of storage, were not detected at all in the samples at 6th and 9th months of storage, and in these samples, only yeasts were observed. We did not isolate any microfungi from malting barley of the Wintmalt variety after 9 months of storage. Strains of Penicillium forming a dominant part of fungal isolates from barley stored in silo were less often isolated from samples stored in floor warehouse, mainly in the Wintmalt variety.
Analysis of the samples of malted barley grain revealed that malting conditions were favorable for fungal growth (
Table 10). Changes in the abundance of fungi in malted barley grain were evident. In the malt from freshly harvested grains, the total yeast count was from 2.2 × 10
3 to 4.5 × 10
3 CFU/g and filamentous fungi from 1.1 × 10
3 to 2.2 × 10
3 CFU/g. We detected 6 genera of filamentous fungi and 3 genera of ascomycetous yeasts and 2 genera of basidiomycetous yeasts from the malting trials.
Aspergillus and
Cladosporium were the dominant genera in the samples of barley after malting.
In this work, the initial fungal counts during storage gradually increased in malt prepared from barley at 3rd, 6th, and 9th month under both types of storage. Alternaria formed a dominant part of filamentous fungal isolates from malted barley grains stored for 9 months in silo, followed by Penicillium. At the end of the 9th month, Mucor dominated in malted barley grains stored in floor warehouse, where Alternaria was the dominant genus at 3 months of storage; at 6th month of storage, there were Alternaria, Penicillium, and Mucor and at 9th month of storage, only Penicillium dominated. Rhodotorula spp. formed a dominant part of yeast isolates in malt prepared from the Laudis and Kangoo varieties and Candida saitoana and Sporobolomyces roseus dominated in malt prepared from the Wintmalt variety throughout the whole storage period in both types of storage.