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Location and extent of fungal mycelium in grains of barley
[ 26 7 ] Trans. Br. mycol. Soc. 56 (2), 267-273 (1971) Printed in Great Britain
LOCATION AND EXTENT OF FUNGAL MYCELIUM IN GRAINS OF BARLEY By D. W. WARNOCK
AND
T. F. PREECE
Department oj Agricultural Sciences, University of Leeds (With Plate 32 and
1
Text-figure)
Cryostat sectioning and phenol-acetic-aniline blue staining was used in a detailed study of the location of the internal fungal mycelium in apparently healthy grains of barley plants. Hyphae were observed in the parenchyma layer of the lemma and palea which enclose the mature caryopsis, and within the pericarp layer of the caryopsis itself. No mycelium was observed in other parts of the grain. As assessment technique is described for the estimation of fungal mycelium present within individual grains.
Christensen (1957) and Mulinge & Chesters (1970) have described the use of plate culture techniques for the isolation and estimation of total fungi present in and on grains of cereal plants. The results of these techniques are dependent on the selectivity of the agar medium used and temperature and length of incubation. These techniques do not give an assessment of the amount of internal mycelium that is present and potentially capable of toxin production. Hyde (1950) described an assessment technique for estimating the subepidermal fungal mycelium in grains of wheat but there have been no reports of similar techniques for barley. Tuite & Christensen (1955) embedded, sectioned and stained grains of barley. They noted' abundant fungal mycelium' in the parenchyma layer of the lemma and palea but gave no details. Mycelium was also present, 'but less abundant', in the pericarp layer. In the hulled barleys grown in Britain, the mature caryopsis is enclosed within the lemma and palea; the lemma extends some two-thirds of the way round, its margins overlapping those of the palea. Except at the apex and on the edges, the lemma and palea are divisible into four layers: (I) An outer epidermis consisting of elongated 'wavy' cells, small 'twin' cells and small' round' cells. (2) A layer of sclerenchyma fibres which are either thick- or thin-walled. (3) A parenchyma layer consisting of characteristic irregularly square or quadrilateral cells, through which run the fibro-vascular bundles. (4) An inner epidermis consisting of more or less polygonal cells and characteristic trichomes and stomata. The lemma and palea average 13 % of the whole grain (Kent, 1943). The inner epidermis of the lemma and palea is fused to the epicarp. The pericarp consists of a single layer of longitudinally elongated epicarp cells and several layers of similar mesocarp cells. These cells are very compressed in cross-section, cannot easily be distinguished, and together will be considered as (5) the pericarp layer. 17-2
268
Transactions British Mycological Society
A double cross-cell layer consisting of transversely elongated cells and a few detached tube cells are occasionally observed in cross-section. The endosperm of the barley grain averages 73 % of the whole grain (Kent, 1943) and is enclosed by the layers described above. It consists of (6) two to four layers of aleurone cells, which are more or less rectangular or polygonal in shape with very thick walls and dense contents, which surround (7) the starch-containing cells. The object of the present work was to study the distribution of the internal fungal mycelium and to attempt to develop a method for its assessment. MATERIALS AND METHODS
A sample of barley grains which had been involved in an outbreak of kidney disease in calves was studied. The sample was not visibly mouldy. The percentage frequencies of the various fungi present were estimated by incubating surface-sterilized grains on Oxoid potato-dextrose agar plates at 25° for 1 week. Those isolated were predominantly 'storage fungi' and included Aspergillus sp. (5 %), Mucor sp. (15 %) and Penicillium sp. (99 %). The grains to be sectioned were immersed for several hours in a 5 % aqueous solution of polyvinyl alcohol (MO 5/140 from Bush, Beach and Segner Bayley Ltd) to facilitate sectioning and reduce freezing damage. Each grain was mounted directly upon a metal block holder from the microtome. The block holder, precooled to - 30° in the cryostat cabinet, was clamped on a 'fast freeze' unit (Slee Medical Equipment Ltd, London) attached to a carbon dioxide cylinder and further cooled to -60°. A small drop of water was placed on the cooled block holder and as the last part was about to freeze, a grain was placed on the ice, so that it became partly trapped. When the tissue was completely frozen the block holder was transferred to the cryostat cabinet. The cryostat or low-temperature microtome cabinet (Bright Instrument Ltd, Huntingdon, England), consisted of a modified Cambridge rocking microtome positioned in a refrigerated cabinet and fitted with external controls. Section cutting was controlled visually through a window in the front of the cabinet. Serial longitudinal sections were cut at IO/Lm thickness at - 25 to - 30°. Sections were transferred to prepared slides and floated with distilled water to expand the tissue. The slides were dried and stored to await staining. The sections were immersed for 1 h in phenol-acetic-aniline blue, rinsed in 95 % ethyl alcohol, followed by a quick rinse in Euparal essence and then mounted in Euparal (Jones & Mollison, 1948). They were examined at x 400 magnification using a Patholux microscope (Vickers Ltd, York, England). RESULTS
A series of fifty longitudinal sections were examined in detail from each barley grain. Each observation of an individual fungal hypha or separate aggregate of fungal hyphae was termed a 'hyphal unit'. The amount of mycelium present was estimated by counting the number of hyphal units
Fungi in barley grains. D. W. Warnock and T. F. Preece 269 present in the different histological layers of each section. Some of the observations on a typical grain are set out in Table I . The mean number of hyphal units present in the 50 sections of this grain were: outer epidermis 0, sclerenchyma 0, parenchyma layer 14'5, inner epid ermis 0, peri carp layer 14, aleurone cell layer 0, starch cells 0. Table I. N umber of hyphal units observed in each of the different layers of a typical series of 10 pm longitudinal sections of an individual barley grain Seri al sec tion no. 13 14 IS 16 17
Outer epidermis ( I)
Sclerenchy ma (2)
Par enchyma layer (3)
0
0
16 24 19 18
0
0
II
0
0
0
0 0
0
15
Inner epidermis (4)
Peri ca rp layer (S)
Aleu rone cells (6)
0
21 23 13 20 16
0
0
0 0 0 0
0 0 0 0
0 0 0 0
13
14
16
17
Starch cells (7)
T ext-fig. I . Fungal m ycelium in a barley gr ain: location of th e hyphal units observed in the various histological region s of th e five 10 J1-rn longitudinal sections listed in Table I.
Transactions British Mycological Society
270
The actual location of the hyphal units recorded in the different histological layers of the five sections listed in Table 1 is illustrated in Text-fig. 1. Fungal mycelium was observed only in the parenchyma layer (Pi. 32 fig. 1) of the lemma and palea and in the pericarp layer (PI. 32 fig. 2) of the caryopsis itself. Using the technique of counting the number of hypha I units observed in each layer of a series of longitudinal sections, the following simplified grading scale was developed to describe the amount of mycelium present in sections of individual grains. The scale is divided into three classes: Class 0, no visible infection, no hyphal units observed . Class I, limitedinfection, a maximum of ten hyphal units observed in each histological layer of individual sections. Class 2, extensive infection, more than ten hyphal units observed in each layer. The use of this simplified grading scale enabled the pattern of distribution and extent of the internal mycelium in a number of grains to be described more rapidly. As a routine pro cedure, 100 serial longitudinal sections were pro duced from each grain and twenty of these sections were examined and graded. Since each section was 10 !tm thick, the total depth of the grain sampled was 1 mm. Typical results from one grain are set out in Table 2. Table 2 . Assessment offungal mycelium present in the various layers tif twenry 10 um thick serial longitudinal sections tif one barley grain using a grading scale Serial section no.
(0,
Parenchyrna layer
Pericarp layer
All other layers
Serial section no.
51 56 61 66 71 76 81 86 91 96
I
2
2
6
2
2
II
I
2
0 0 0
16 21 26 31 36 41 46
2
2
0
2
2
2
2
2 2
0 0
0
0
I
I I
I
No visible mycelium;
I,
0 0
0 0
maximum of
IO
hyphal units;
Parenchyma layer
Pericarp layer
0 0 0 0 0 0 0 0 0 0
I
2 0
I
I
2
I
I
I
0
I
I
I
I
I
2,
0
I
I
more than
All other layers
10
hyphal units.)
The data shown in Table 2 may be summarized: parenchyma layer, Class 0 recorded from two sections, Class 1 recorded from twelve sections, Class 2 recorded from six sections. Pericarp layer : Class 0 recorded from three sections, Class 1 recorded from eight sections, Class 2 recorded from nine sections. All other layers: Class 0 recorded from twenty sections. These assessment summaries from ten typical grains are set out in Table 3. Having assessed the amounts of mycelium in the various layers of individual grains (Table 3), a single grain assessment figure was then derived. The method of derivation of this single figure for one grain (grain 3 in Table 3) is shown in Table 4. In calculating the single grain assessment figure, the Class 2 figures were multiplied by 2 thus making an allowance for the number of hyphal units
Fungi in barley grains. D. W. Warnock and T. F. Preece
271
present. The single grain assessment figures for the sample of ten grains (A I-A 10) shown in Table 3 are set out in Table 5 together with assessment figures from another sample of ten barley grains (B I-B 10) for comparison. These ten grains were from a sample received from the N.A.A.S. Trawscoed, Wales. Table 3. Assessments offungal mycelium present in the various layers of ten barley grains Parenchyma layer Pericarp layer All other layers ,--~-----, , '------~, Grain ~---, no, Class 0 Class I Class 2 Class 0 Class I Class 2 Class 0 Class I Class 2 I 2 3 4 5 6 7 8 9 10
2 0 0 2 5 0 0 4 1 0
14 II 3 I7 15 15 14 13 14 12
8 8 2 II 12 13 16 15 14 9
3
4 9 17 1 0 5 6 3 5 8
0 0
8 4 1 0
3 2 0
20 20 20 20 20 20 20 20 20 20
9 12 18 4 6 4 2 4 II
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0.
0
Table 4. Derivation of a single assessment figure for the amount offungal mycelium present in one barley grain Parenchyma layer
Score for extent of mycelium Calculation of assessment
Class 0
Class I
Class 2
0
3
17
0
+
3
+
Pericarp layer
,
A
I
34
"
I
Class 0
Class I
0
+
0
18
2
+
2
, Class 2
+
36=75
Table 5. Comparison of individual grain assessment figures from two samples of barley grains Grain no. AI A2 A3 A4 A5 A6 A7 A8 A9 AIO
Assessment figure 48 61 75 32 35 50 5° 38 46 59 Mean 49'4
Grain no.
Assessment figure
BI B2 B3 B4 B5 B6 B7 B8 B9 BIO
29 51 47 46 57 55 23 8 30 I Mean 34'7
Thus the assessment procedure for a grain sample is as follows. A random sample of ten grains is sectioned, twenty stained sections of each grain examined and the extent of fungal mycelium in the different layers scored as absent (0), limited (I) or extensive (2). Single grain assessment figures and a mean assessment figure for the sample of 10 grains are then calculated.
272
Transactions British Mycological Society DISCUSSION
The internal fungal mycelium of the apparently healthy barley grains examined was present to an approximately equal extent in the parenchyma layer of the lemma and palea which enclose the mature caryopsis and in the pericarp layer of the caryopsis itself. The other layers and the embryos appear to be free from hyphae. Thus over 70 % of each grain was uninfected. The choice of techniques to study the fungi present in cereal grains is very important. In spite of their limitations, plate-culture techniques are usually adopted in large-scale studies. The occurrence of different fungal species can be estimated by the direct plating technique (Christensen, 1957). Among the range of fungi isolated from barley grains using this technique are a number of species of medical and veterinary importance including Absidia corymbifera, Aspergillusfumigatus, Mucor pusillus and Thermoactinomyces vulgaris (Flannigan, 1969). Quantitative studies on the fungi present in the cereal grains have been made almost exclusively by the dilution plate technique (Christensen, 1957). The range of fungi isolated during dilution plate studies includes a high proportion of heavily sporulating species which tend to be overestimated, while purely mycelial species and weakly sporulating species are underestimated. Another experimental approach which has been used to estimate the fungal population is that described by Lacey (1965) in which the atmospheric spore count is taken as a reflection of the fungal species present within grains. The main disadvantage of this technique also arises from overestimation of heavily sporulating species. In the three techniques described above, the interpretation of experimental results to give an assessment of the amount of mycelium present within the grain is not possible. The object of the present work was to observe the location of the internal mycelium directly and to produce comparative figures to describe the amount present. Although the technique now described still does not give an absolute measurement of the mycelium, such absolute measurement will be very difficult to derive. It will almost certainly depend on advanced biochemical techniques; for example most fungi, unlike higher plants, have chitin in their walls, which breaks down to glucosamine on acid hydrolysis. Estimation of glucosamine may perhaps give a quantitative estimate of the amount of fungal tissue present in the infected plant (Ride & Drysdale, 1969). It can be seen from Table 5 that the assessment figures for two different samples of barley grain are similar. However, this does not mean that the species present are the same, nor need they have the same potential for toxin production. Specific identification of the hyphae present, and therefore comparison with plate-culture results, is not possible using the simple staining technique described here. Studies using immunologically specific antisera labelled with tracer fluorescent dyes are now in progress. We have not examined grains for actinomycetes, nor have we examined visibly moulded grains. Both these aspects need further study.
Trans. Br, mycol. Soc.
Vol. 56.
Plate 32
OE
(Facing p. 273)
Fungi in barleygrains. D. W. Warnock and T. F. Preece 273 We are grateful to Professor J. H. Western for his advice during the course of this work. We should like to thank the Home Grown Cereals Authority for the provision of a postgraduate studentship for D. W. W. and also Dr L. V. Evans for his assistance with the Plate. REFERENCES
CHRISTENSEN, C. M. (1957). Deterioration of stored grains by fungi. Botanical Review 23, 108- 1 34. FLANNIGAN, B. (1969). Microflora of dried barley grain. These Transactions, 53, 371-379. HYDE, M. B. (1950). The subepidermal fungi of cereal grains. I. A survey of the world distribution of fungal mycelium in wheat. Annals of Applied Biology 37, 179-186. JONES, P. C. T. & MOLLISON,]. E. (1948). A technique for the quantitative estimation soil micro-organisms. Journal of General Microbiology 2, 54-69. KENT, N. L. (1943)' Some characteristics of barley. Milling 101, 240-242. LACEY,]. (1965). Interim report on the microflora of airborne dust from moist-stored barley grain. In Experimental farm buildings report no. 4. Agricultural Research Council. MULINGE, S. K. & CHESTERS, C. G. C. (1970). Ecology of fungi associated with moist stored barley grain. Annals of Applied Biology 65, 277-284. RIDE, ]. P. & DRYSDALE, R. B. (1969). The chemical estimation of fungus in plant tissue. Journal of General Microbiology 59, viii (Proceedings). TUITE,]. F. & CHRISTENSEN, C. M. (1955). Grain storage studies 16. Influence of storage conditions upon the fungus flora of barley seed. Cereal Chemistry 32, 1-11.
EXPLANATION OF PLATE
32
Location of fungal hyphae in the different histological layers of barley grains. Fig. I. Septate mycelium in the parenchyma layer of the lemma or palea. Fig. 2. Septate mycelium in the pericarp layer of the caryopsis. DE, Outer epidermis; Sc, sclerenchyma fibres; Pa, parenchyma layer; IE, inner epidermis; Pe, pericarp layer.
(Accepted for publication 5 November 1970)
LOCATION AND EXTENT OF FUNGAL MYCELIUM IN GRAINS OF BARLEY By D. W. WARNOCK
AND
T. F. PREECE
Department oj Agricultural Sciences, University of Leeds (With Plate 32 and
1
Text-figure)
Cryostat sectioning and phenol-acetic-aniline blue staining was used in a detailed study of the location of the internal fungal mycelium in apparently healthy grains of barley plants. Hyphae were observed in the parenchyma layer of the lemma and palea which enclose the mature caryopsis, and within the pericarp layer of the caryopsis itself. No mycelium was observed in other parts of the grain. As assessment technique is described for the estimation of fungal mycelium present within individual grains.
Christensen (1957) and Mulinge & Chesters (1970) have described the use of plate culture techniques for the isolation and estimation of total fungi present in and on grains of cereal plants. The results of these techniques are dependent on the selectivity of the agar medium used and temperature and length of incubation. These techniques do not give an assessment of the amount of internal mycelium that is present and potentially capable of toxin production. Hyde (1950) described an assessment technique for estimating the subepidermal fungal mycelium in grains of wheat but there have been no reports of similar techniques for barley. Tuite & Christensen (1955) embedded, sectioned and stained grains of barley. They noted' abundant fungal mycelium' in the parenchyma layer of the lemma and palea but gave no details. Mycelium was also present, 'but less abundant', in the pericarp layer. In the hulled barleys grown in Britain, the mature caryopsis is enclosed within the lemma and palea; the lemma extends some two-thirds of the way round, its margins overlapping those of the palea. Except at the apex and on the edges, the lemma and palea are divisible into four layers: (I) An outer epidermis consisting of elongated 'wavy' cells, small 'twin' cells and small' round' cells. (2) A layer of sclerenchyma fibres which are either thick- or thin-walled. (3) A parenchyma layer consisting of characteristic irregularly square or quadrilateral cells, through which run the fibro-vascular bundles. (4) An inner epidermis consisting of more or less polygonal cells and characteristic trichomes and stomata. The lemma and palea average 13 % of the whole grain (Kent, 1943). The inner epidermis of the lemma and palea is fused to the epicarp. The pericarp consists of a single layer of longitudinally elongated epicarp cells and several layers of similar mesocarp cells. These cells are very compressed in cross-section, cannot easily be distinguished, and together will be considered as (5) the pericarp layer. 17-2
268
Transactions British Mycological Society
A double cross-cell layer consisting of transversely elongated cells and a few detached tube cells are occasionally observed in cross-section. The endosperm of the barley grain averages 73 % of the whole grain (Kent, 1943) and is enclosed by the layers described above. It consists of (6) two to four layers of aleurone cells, which are more or less rectangular or polygonal in shape with very thick walls and dense contents, which surround (7) the starch-containing cells. The object of the present work was to study the distribution of the internal fungal mycelium and to attempt to develop a method for its assessment. MATERIALS AND METHODS
A sample of barley grains which had been involved in an outbreak of kidney disease in calves was studied. The sample was not visibly mouldy. The percentage frequencies of the various fungi present were estimated by incubating surface-sterilized grains on Oxoid potato-dextrose agar plates at 25° for 1 week. Those isolated were predominantly 'storage fungi' and included Aspergillus sp. (5 %), Mucor sp. (15 %) and Penicillium sp. (99 %). The grains to be sectioned were immersed for several hours in a 5 % aqueous solution of polyvinyl alcohol (MO 5/140 from Bush, Beach and Segner Bayley Ltd) to facilitate sectioning and reduce freezing damage. Each grain was mounted directly upon a metal block holder from the microtome. The block holder, precooled to - 30° in the cryostat cabinet, was clamped on a 'fast freeze' unit (Slee Medical Equipment Ltd, London) attached to a carbon dioxide cylinder and further cooled to -60°. A small drop of water was placed on the cooled block holder and as the last part was about to freeze, a grain was placed on the ice, so that it became partly trapped. When the tissue was completely frozen the block holder was transferred to the cryostat cabinet. The cryostat or low-temperature microtome cabinet (Bright Instrument Ltd, Huntingdon, England), consisted of a modified Cambridge rocking microtome positioned in a refrigerated cabinet and fitted with external controls. Section cutting was controlled visually through a window in the front of the cabinet. Serial longitudinal sections were cut at IO/Lm thickness at - 25 to - 30°. Sections were transferred to prepared slides and floated with distilled water to expand the tissue. The slides were dried and stored to await staining. The sections were immersed for 1 h in phenol-acetic-aniline blue, rinsed in 95 % ethyl alcohol, followed by a quick rinse in Euparal essence and then mounted in Euparal (Jones & Mollison, 1948). They were examined at x 400 magnification using a Patholux microscope (Vickers Ltd, York, England). RESULTS
A series of fifty longitudinal sections were examined in detail from each barley grain. Each observation of an individual fungal hypha or separate aggregate of fungal hyphae was termed a 'hyphal unit'. The amount of mycelium present was estimated by counting the number of hyphal units
Fungi in barley grains. D. W. Warnock and T. F. Preece 269 present in the different histological layers of each section. Some of the observations on a typical grain are set out in Table I . The mean number of hyphal units present in the 50 sections of this grain were: outer epidermis 0, sclerenchyma 0, parenchyma layer 14'5, inner epid ermis 0, peri carp layer 14, aleurone cell layer 0, starch cells 0. Table I. N umber of hyphal units observed in each of the different layers of a typical series of 10 pm longitudinal sections of an individual barley grain Seri al sec tion no. 13 14 IS 16 17
Outer epidermis ( I)
Sclerenchy ma (2)
Par enchyma layer (3)
0
0
16 24 19 18
0
0
II
0
0
0
0 0
0
15
Inner epidermis (4)
Peri ca rp layer (S)
Aleu rone cells (6)
0
21 23 13 20 16
0
0
0 0 0 0
0 0 0 0
0 0 0 0
13
14
16
17
Starch cells (7)
T ext-fig. I . Fungal m ycelium in a barley gr ain: location of th e hyphal units observed in the various histological region s of th e five 10 J1-rn longitudinal sections listed in Table I.
Transactions British Mycological Society
270
The actual location of the hyphal units recorded in the different histological layers of the five sections listed in Table 1 is illustrated in Text-fig. 1. Fungal mycelium was observed only in the parenchyma layer (Pi. 32 fig. 1) of the lemma and palea and in the pericarp layer (PI. 32 fig. 2) of the caryopsis itself. Using the technique of counting the number of hypha I units observed in each layer of a series of longitudinal sections, the following simplified grading scale was developed to describe the amount of mycelium present in sections of individual grains. The scale is divided into three classes: Class 0, no visible infection, no hyphal units observed . Class I, limitedinfection, a maximum of ten hyphal units observed in each histological layer of individual sections. Class 2, extensive infection, more than ten hyphal units observed in each layer. The use of this simplified grading scale enabled the pattern of distribution and extent of the internal mycelium in a number of grains to be described more rapidly. As a routine pro cedure, 100 serial longitudinal sections were pro duced from each grain and twenty of these sections were examined and graded. Since each section was 10 !tm thick, the total depth of the grain sampled was 1 mm. Typical results from one grain are set out in Table 2. Table 2 . Assessment offungal mycelium present in the various layers tif twenry 10 um thick serial longitudinal sections tif one barley grain using a grading scale Serial section no.
(0,
Parenchyrna layer
Pericarp layer
All other layers
Serial section no.
51 56 61 66 71 76 81 86 91 96
I
2
2
6
2
2
II
I
2
0 0 0
16 21 26 31 36 41 46
2
2
0
2
2
2
2
2 2
0 0
0
0
I
I I
I
No visible mycelium;
I,
0 0
0 0
maximum of
IO
hyphal units;
Parenchyma layer
Pericarp layer
0 0 0 0 0 0 0 0 0 0
I
2 0
I
I
2
I
I
I
0
I
I
I
I
I
2,
0
I
I
more than
All other layers
10
hyphal units.)
The data shown in Table 2 may be summarized: parenchyma layer, Class 0 recorded from two sections, Class 1 recorded from twelve sections, Class 2 recorded from six sections. Pericarp layer : Class 0 recorded from three sections, Class 1 recorded from eight sections, Class 2 recorded from nine sections. All other layers: Class 0 recorded from twenty sections. These assessment summaries from ten typical grains are set out in Table 3. Having assessed the amounts of mycelium in the various layers of individual grains (Table 3), a single grain assessment figure was then derived. The method of derivation of this single figure for one grain (grain 3 in Table 3) is shown in Table 4. In calculating the single grain assessment figure, the Class 2 figures were multiplied by 2 thus making an allowance for the number of hyphal units
Fungi in barley grains. D. W. Warnock and T. F. Preece
271
present. The single grain assessment figures for the sample of ten grains (A I-A 10) shown in Table 3 are set out in Table 5 together with assessment figures from another sample of ten barley grains (B I-B 10) for comparison. These ten grains were from a sample received from the N.A.A.S. Trawscoed, Wales. Table 3. Assessments offungal mycelium present in the various layers of ten barley grains Parenchyma layer Pericarp layer All other layers ,--~-----, , '------~, Grain ~---, no, Class 0 Class I Class 2 Class 0 Class I Class 2 Class 0 Class I Class 2 I 2 3 4 5 6 7 8 9 10
2 0 0 2 5 0 0 4 1 0
14 II 3 I7 15 15 14 13 14 12
8 8 2 II 12 13 16 15 14 9
3
4 9 17 1 0 5 6 3 5 8
0 0
8 4 1 0
3 2 0
20 20 20 20 20 20 20 20 20 20
9 12 18 4 6 4 2 4 II
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0.
0
Table 4. Derivation of a single assessment figure for the amount offungal mycelium present in one barley grain Parenchyma layer
Score for extent of mycelium Calculation of assessment
Class 0
Class I
Class 2
0
3
17
0
+
3
+
Pericarp layer
,
A
I
34
"
I
Class 0
Class I
0
+
0
18
2
+
2
, Class 2
+
36=75
Table 5. Comparison of individual grain assessment figures from two samples of barley grains Grain no. AI A2 A3 A4 A5 A6 A7 A8 A9 AIO
Assessment figure 48 61 75 32 35 50 5° 38 46 59 Mean 49'4
Grain no.
Assessment figure
BI B2 B3 B4 B5 B6 B7 B8 B9 BIO
29 51 47 46 57 55 23 8 30 I Mean 34'7
Thus the assessment procedure for a grain sample is as follows. A random sample of ten grains is sectioned, twenty stained sections of each grain examined and the extent of fungal mycelium in the different layers scored as absent (0), limited (I) or extensive (2). Single grain assessment figures and a mean assessment figure for the sample of 10 grains are then calculated.
272
Transactions British Mycological Society DISCUSSION
The internal fungal mycelium of the apparently healthy barley grains examined was present to an approximately equal extent in the parenchyma layer of the lemma and palea which enclose the mature caryopsis and in the pericarp layer of the caryopsis itself. The other layers and the embryos appear to be free from hyphae. Thus over 70 % of each grain was uninfected. The choice of techniques to study the fungi present in cereal grains is very important. In spite of their limitations, plate-culture techniques are usually adopted in large-scale studies. The occurrence of different fungal species can be estimated by the direct plating technique (Christensen, 1957). Among the range of fungi isolated from barley grains using this technique are a number of species of medical and veterinary importance including Absidia corymbifera, Aspergillusfumigatus, Mucor pusillus and Thermoactinomyces vulgaris (Flannigan, 1969). Quantitative studies on the fungi present in the cereal grains have been made almost exclusively by the dilution plate technique (Christensen, 1957). The range of fungi isolated during dilution plate studies includes a high proportion of heavily sporulating species which tend to be overestimated, while purely mycelial species and weakly sporulating species are underestimated. Another experimental approach which has been used to estimate the fungal population is that described by Lacey (1965) in which the atmospheric spore count is taken as a reflection of the fungal species present within grains. The main disadvantage of this technique also arises from overestimation of heavily sporulating species. In the three techniques described above, the interpretation of experimental results to give an assessment of the amount of mycelium present within the grain is not possible. The object of the present work was to observe the location of the internal mycelium directly and to produce comparative figures to describe the amount present. Although the technique now described still does not give an absolute measurement of the mycelium, such absolute measurement will be very difficult to derive. It will almost certainly depend on advanced biochemical techniques; for example most fungi, unlike higher plants, have chitin in their walls, which breaks down to glucosamine on acid hydrolysis. Estimation of glucosamine may perhaps give a quantitative estimate of the amount of fungal tissue present in the infected plant (Ride & Drysdale, 1969). It can be seen from Table 5 that the assessment figures for two different samples of barley grain are similar. However, this does not mean that the species present are the same, nor need they have the same potential for toxin production. Specific identification of the hyphae present, and therefore comparison with plate-culture results, is not possible using the simple staining technique described here. Studies using immunologically specific antisera labelled with tracer fluorescent dyes are now in progress. We have not examined grains for actinomycetes, nor have we examined visibly moulded grains. Both these aspects need further study.
Trans. Br, mycol. Soc.
Vol. 56.
Plate 32
OE
(Facing p. 273)
Fungi in barleygrains. D. W. Warnock and T. F. Preece 273 We are grateful to Professor J. H. Western for his advice during the course of this work. We should like to thank the Home Grown Cereals Authority for the provision of a postgraduate studentship for D. W. W. and also Dr L. V. Evans for his assistance with the Plate. REFERENCES
CHRISTENSEN, C. M. (1957). Deterioration of stored grains by fungi. Botanical Review 23, 108- 1 34. FLANNIGAN, B. (1969). Microflora of dried barley grain. These Transactions, 53, 371-379. HYDE, M. B. (1950). The subepidermal fungi of cereal grains. I. A survey of the world distribution of fungal mycelium in wheat. Annals of Applied Biology 37, 179-186. JONES, P. C. T. & MOLLISON,]. E. (1948). A technique for the quantitative estimation soil micro-organisms. Journal of General Microbiology 2, 54-69. KENT, N. L. (1943)' Some characteristics of barley. Milling 101, 240-242. LACEY,]. (1965). Interim report on the microflora of airborne dust from moist-stored barley grain. In Experimental farm buildings report no. 4. Agricultural Research Council. MULINGE, S. K. & CHESTERS, C. G. C. (1970). Ecology of fungi associated with moist stored barley grain. Annals of Applied Biology 65, 277-284. RIDE, ]. P. & DRYSDALE, R. B. (1969). The chemical estimation of fungus in plant tissue. Journal of General Microbiology 59, viii (Proceedings). TUITE,]. F. & CHRISTENSEN, C. M. (1955). Grain storage studies 16. Influence of storage conditions upon the fungus flora of barley seed. Cereal Chemistry 32, 1-11.
EXPLANATION OF PLATE
32
Location of fungal hyphae in the different histological layers of barley grains. Fig. I. Septate mycelium in the parenchyma layer of the lemma or palea. Fig. 2. Septate mycelium in the pericarp layer of the caryopsis. DE, Outer epidermis; Sc, sclerenchyma fibres; Pa, parenchyma layer; IE, inner epidermis; Pe, pericarp layer.
(Accepted for publication 5 November 1970)