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Some influences of storage fungi, temperatures and relative humidity on the germinability of grass seeds
3. stored Prod.
Res., 1967,
Vol. 3, pp. 335-343.
Pergamon Press Ltd.
Printed in Great Britain.
Some Influences of Storage Fungi, Temperatures and Relative Humidity on the Germinability of Grass Seeds MARTIN
M. KULIK and OREN
L. JUSTICE
Market Quality Research Division, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705, U.S.A. (First receiued 20 February, 1967, and infinal form 30
June,1967)
Abstract--Seeds of Poa pratensis, Festuca rubra, F. arundinacea, and Lolium multz@um were storedat 10, 20, 30, or 35”C, and at 35, 55, 75, or 95 per cent r.h. for up to 12 months. Samples stored at 30°C and 95 per cent r.h. and 35°C and 75 or 95 per cent r.h. de-creased in germinability after 2 months of storage. At 20°C and either 75 or 95 per cent r.h., seeds decreased more slowly in germinative ability than at the higher temperatures but this decrease was appreciable. Seeds stored at 10°C and at 35, 55, 75, or 95 per cent r.h. germinated well after 12 months. Equilibrium moisture content of the seeds after storage at 35 to 75 per cent r.h. for 2, 4, and 8 months varied little among the four species at each relative humidity level and was not affected by temperature in the range of lO-35°C. Fungi of the AspergiUuv glaucus group could not always be isolated from large numbers of seeds in lots that had declined in germinability. Seeds of all four grasses apparently carried superficially-borne species of the A. glaucus group (primarily A. amstefodami) in a ‘dormant’ atate at harvest. Members of this group were isolated from substantial numbers of seeds that had been stored for 3 months at 30°C and 75 per cent r.h. These fungi were not isolated from seeds stored for the same length of time at 30°C and 35 per cent r.h. ; however, field fungi grew from many of these seeds. INTRODUCTION
STORED seeds are subject to deterioration due to fungi, normal physiological processes in the seeds, insect pests, and rodents. CHRISTENSEN (1957), TUITE and CHRISTENSEN (1957), and CHRISTENSEN and LOPEZ (1963), and others, have shown that certain types of moulds, the so-called storage fungi, can invade and destroy cereals and other seeds when storage conditions become favourable for fungal growth. These fungi, mainly members of the genera As#ergillus and Penicillium, and especially species in the Aspergillus glaucus group,* can grow under relatively dry conditions. Thus, these fungi must be taken into consideration whenever seeds are stored. * Hereafter the expression “A. A. glaucus glaucus group (RAPER and FENNELL, 1965).
spp.” will be used to denote species of the Aspergihs
335
336
kfARTIN
hi. KULIK and OREN L.
JUSTICE
A number of factors, including the growth of suburban areas, have contributed to an increased demand for lawn grasses in the United States, leading to larger quantities of these seeds in market channels and in storage. It is important to know the role which storage fungi play in grass seed deterioration, as well as the effects of storage conditions on seed germinability. iVe undertook to identify the storage fungi associated with certain grass seeds and to determine the influence of fungi, as well as temperature and r.h. on the maintenance of high germination after storage. MATERIALS
AND METHODS
At the beginning of this investigation, a study was undertaken to determine the species and numbers of storage fungi associated with grass seeds. Commercially cleaned seeds of Poa pratensis L., Festuca rubra L., Festuca arundinacea Schreb., and Lolium multzjlorum Lam. were obtained from a number of seed companies. Seeds harvested in 1963 were secured in 1964 after commercial storage for one year. Although the storage histories of these samples were not available, they probably represented a typical cross-section of carry-over grass seed stocks. In addition, samples of seeds produced in 1964 and 1965 were obtained after commercial cleaning, but before storage had started. One hundred seeds from each lot were swirled for 1 min in 1 per cent sodium hypochlorire, rinsed briefly with sterile distilled water, and placed on 10, 15, or 20 per cent NaCl-malt agar. Conidia from developing colonies of fungi were transferred to other Petri dishes for the identification of the isolates. Subsequently, seeds of the four above-mentioned species, intended for use in storage studies, were obtained at harvest time from several growers. The seeds were shipped in cloth bags by surface mail and were kept at 22-25°C and 25-40 per cent r.h. while we cleaned them. Trash and light-weight florets were removed with a miniature seed cleaner. To ascertain whether or not storage fungi were present at the beginning of the experiment, seeds from each cleaned sample were placed on 10 per cent NaCl-malt agar without being rinsed first with sodium hypochlorite. Those samples found to be free of storage fungi, as shown by our plating results, were selected for storage studies. Germination and moisture content determinations followed the procedures of JUSTICE (1952) and HART et al. (1959), respectively. At the time the seed was placed in storage, its germination was as follows: Poa pratensis, 89 per cent; Festuca rubra, 96 per cent; F. arundinacea, 95 per cent; and Lolium multiJorum, 98 per cent. Seeds of each species were placed in separate 150 ml glass jars which were then covered with fine cloth gauze to avoid mixing. The jars with the seeds were put into glass desiccators containing saturated salt solutions to produce r.h. of 35, 55, 75, or 95 per cent (O’BRIEN, 1948). The desiccators were placed in incubators held at 10, 20, 30, or 35°C. In this manner, sixteen combinations of r.h. and temperature were maintained. Subsamples were withdrawn after 2, 4, 8, or 12 months. To detect storage fungi, seeds were swirled for 1 min in 1 per cent sodium hypochlorite, followed by a brief rinse with sterile, distilled water, and were then placed on 10 per cent NaCl-malt agar. The plates containing the seeds were incubated at 22-25X. Germination percentages were based on 200 seeds per subsample. Moisture contents (wet weight
Some Influences
of Storage
Fungi on the Germinability
337
of Grass Seeds
basis) were determined at each withdrawal date, except that no moisture determinations were made after 8 months. Two 2-g portions of seeds from each subsample were dried at 130°C for 1 hr in a hot air oven. RESULTS
Fungi associated with grass seedr
A large number of samples of grass seeds from the 1963 carry-over stocks as well as seeds from 1964 and 1965 that had not been stored, were assayed for storage fungi. The results in Table 1 for seeds of Poa pratensis are typical of those obtained from many samples of seeds of Festuca rubra, F. arundinacea, and Lolium mult$orum. The principal storage fungi encountered were A. glaucus spp., with A. amstelodami (Mang.) Thorn and Church most commonly isolated; some other species of Aspergillus were isolated but only in trace amounts (Table 1).
TABLE ON
10
1. per
NUMBER Cent
OF Pea
SODIUM
Year of seed harvestt 1963 1963 1963 1963 1963 1964 1964 1964 1965 1965 1965
pratensis
SEEDS
CHLORIDE-MALT
FROX
WHICH
AGAR
WITHOUT
FUSGI
WERE
SODIC’M
ISOLATED
WHEN
HYPOCHLORITE
Aspergillus glaucus spp.
Penicillium
species
Field fungi:
73 0 0 0 3 0 0 41 0 2 0
3-l 2 7 0 2 0 0 0
24 100 100 100 100 100 100 13 100 100 100
1 1 1
C~‘LTC~RED
TREATMENT*
. * One hundred seeds per sample. 7 Seeds from 1963 were stored commercially for one year prior to receipt; the 1964 and 1965 samples were obtained before storage had occurred. $ Principally Alternaria tenuis auct. sensu Wiltshire and Cladosporium spp. In addition, a few samples contained trace amounts of Aspergillus candidus Link, A. niger V. Tiegham, and A. och?accrrs Wilhelm.
Apparently, the grass seeds we plated were either free of storage fungi or only lightly contaminated; however, an occasional sample was heavily infested. Conversely, field fungi, such as Cladosporium herbarum (Pers.) Link ex Fr. and Alternaria tenuis auct. sensu Wiltshire, were often present in very large numbers; they were sometimes isolated from every seed in a subsample. Surface disinfestation with sodium hypochlorite prior to plating indicated that, in most cases, establishment of field fungi was evidently limited to the seed surface. Hyperosmophilic species, such as Aspergillus halophilicus Christensen, Papavizas, and Benjamin and A. restrictus Smith, were ‘not isolated from grass seeds cultured on 15 or 20 per cent NaCl-malt agar. E
338
~~ARTIN hf. KULIK
Comparison isolated
of seed germination
and OREN L. JUSTICE
with the number of see& from
which A. glaucus spP. were
(Note: The data presented in Table 2 represent an average of the data obtained from the four species of grasses. The individual results were averaged because the performance of the seeds of each grass was quite similar.) A. Seedr storedfor 2 months. No apparent correlation existed between germination and the number of seeds from which A. glaucus spp. were isolated. Seeds of the four species taken from samples stored at 12 of 16 temperature-r.h. conditions germinated no lower than 90 per cent, yet the number of seeds per sample from which A. glauczu spp. were isolated varied from 1 to 99 per cent (Table 2). To further illustrate this disparity between number of seeds yielding these fungi and seed viability, these species were isoIated from 84 per cent of seeds taken from samples stored at 35°C and 75 per cent r.h. that germinated 16 per cent. However, A. glaucus spp. were obtained from 96 per cent of seeds from samples stored at 30°C and 95 per cent r.h. that germinated 53 per cent. 2. PERCENTAGEOF GRASS SEEDS FROM WHICHAspergillus glaucw spp. WERE ISOLATED AND per cent GERMINATION AFIZR STORAGE FOR ‘2-12 months UNDER 16 TEMPERATURE-RELATWE
TABLE
HUMIDITY COMBINATIONS*
Relative humidity 35%
Months in storage 2
Storage temp. (“C)
0I
5 1 47 84
1 5 29 20
91 92 89 85
94 94 97 93
1 2 9 2
90 94 93 88
0 1 1 1
16 3 27 48
10
10 20 30 35
O’ Ge?m.
92 91 77 16
7 99 96 93
92 90 53 1
2 50 100
88 88 16
3 100 95
92 83 0
94 95 91 49
0 95
93 64
7 45
94 4
90 94 73 19
1 66
92 27
1
92
93 94 94 94
6 2 8 31
5 3 50 77
91 88 92 92
1 2 26 7 1 5 1
Freqcency A. glaucus SPP*
SPP.
y(, Germ.
0
Freqzenc y A. glaucus
91 92 93 91
10 20 30 35
10
0’
01
SPP*
SPP.
20 30 35
FreqEenc y A. glaucus
95%
“/b Germ.
“/;, Germ.
20 30 35
12
% Frequency A. glatuus
75%
55%
* Each value represents a mean for the four species: Poa pratcnsk, Fcstuca rubra, F’. arundinacea, and Lo&urn muft;POrum. One hundred seeds per sample were cultured on 10 per cent NaCl-malt agar after sodium hypochlorite treatment, and 200 seeds per sample were used to determine germination.
Some Influences of Storage Fungi on the Germinability of Grass Seeds
339
After 2 months, a decline in germination was noted for seed stored at combinations of 30 and 35°C and 75 and 95 per cent r.h. (Table 2). Samples of very low germinability were omitted from this invesbgation after the evaluation at 2 months was completed, and also at 4 and 8 months. B. Seeds storedfor 4 months. Germinability remained high at 10 of the 14 Icvels of temperature and r.h., i.e. 88 per cent or better. The trend toward loss in germination at higher levels of temperature and r,h. continued with a slight loss in germination in seeds stored at 35°C and 55 per cent r.h. (down from 9 1 to 85 per cent after 2 months). Great variation was observed in the number of seeds from which A. gluucus spp. were isolated. This ranged from 1 to 100 per cent with a germination level no lower than 83 per cent in 12 out of 14 samples. C. See& stored for 8 months. Seeds stored under 9 of the 12 conditions germinated 91 per cent or higher. The number of high germinating seeds from which A. glaucus spp. were isolated dropped considerably, i.e. the range was now 0 to 26 per cent compared to 1 to 77 per cent at 4 months. Germination continued to decrease at the higher temperatures and r.h., dropping 42 per cent at 35°C and 55 per cent r.h., 27 per cent at 20°C and 75 per cent r.h., and 95 per cent at 20°C and 95 per cent r.h. (Table 2). D. Seeds stored for 12 months. Almost no A. glaucus spp. were isolated from seeds stored at 10 of the 11 conditions and germination of seeds from 8 of the 11 conditions was no lower than 88 per cent. Germination of lots stored at the higher temperatures and r.h. continued to decrease. For example, decreases between the 8-month and 12-month storage periods were 20 per cent at 30°C and 55 per cent r.h., 61 per cent at 35°C and 55 per cent r.h., and 58 per cent at 20°C and 75 per cent r.h. (Table 2). Curiously, at 20°C and 75 per cent r.h. the percentage frequency of A. glaucus spp. had decreased from 95 to 66 within the 4-month period, although the germination had dropped from 64 to 27 per cent. Moisture
content of seeds at d$erent
temperature-relative
humidity levels
Moisture contents of seeds of the four species studied were determined on a wet weight basis after 2, 4, and 8 months of storage (Table 3). The raw data (not presented) indicated that temperature over a range of lo-35°C had little effect on the moisture content of seeds stored at 35-75 per cent r.h. Moisture contents of seeds of the four grasses used in this investigation varied no more than I per cent among species at a given r.h., except for F. yubra at 95 per cent r.h. (Table 3). These moisture contents remained unchanged during the 8-month storage period. Isolation of A. glaucus plating methods
spp.ji'om seeds apparently ‘free’ of storagejilngi
according to standard
A. glaucus spp. were subsequently isolated from seeds in certain samples that previously had been found ‘free’ of storage fungi by the agar plating procedure. To gain some insight into the cause of this phenomenon, grass seed lots were assayed in 1964 and 1965 for the presence of storage fungi and those found to be ‘free’ of them were stored at 35 or 75 per cent r.h. at 30°C. After 3 months of storage, these seeds were assayed in 10 per cent NaCl-malt agar. The results for each year are summarized as follows: (1) A. gluucus spp, were not isolated from seeds taken from
&fARTIN xi. I(ULIK and OREN
340
TABLE
L.JUSTICE
3. AVERAGE per cent MOISTURE (WET WEIGHT BASIS)OF GRASS SEEDS STORED AT SIXTEEN TEUPERATURE-RELATIVE HUMIDITY COMBINATIONS*
Moisture content/relative humidity Species of grass Poa prateffsis Festka rubra Festuca arundinacea Ldium muftfJ?orfmf
350,;
55:,;
75?,
95”0
8.4
IO-3 10.9 11.0 11.3
13.5 14.1 13.8 14.2
17.3 20.3 18.3 18.1
8.8 9-l
9.1
* Data represent the combined average moisture contents of samples stored at 10, 20, 30, or 35°C and measured at intervals of 2, 4, or 8 months.
samples stored for 3 months at 30°C and 35 per cent r.h., but field fungi were isolated from every seed in almost all samples (Table 4). (2) All seeds from samples stored at 30°C and 75 per cent r.h. for 3 months yielded A. glaucus spp. but no field fungi (Table 4). TABLE 4. INFLUENCE OF STORAGE ENVIROSMENT ON THE NUMBER OF GRASS SEEDS GROWN IN 1964 AND 1965 FROM WHICH Aspergillw glaucuJ spp. AND FIELD FUNGI WERE ISOLATED A. glaucus spp.
Species of grass Poa pratensis
Festuca rubra
No. of samples averaged 5
3
Festuca ar ffndinacea
2
Lolifim JnJfLtJgforuJJl
5
Tb r.h. of storage*
Total seedborne populationt 0
35 75
100
35 75
100
35 75
100
35 75
100
0
0
0
Internallyborne populationS
Field fungi Total seedborne populationf
Internallyborne population:
0 33
98 0
11 0
0 37
100 0
26 0
0 62
100 0
33 0
0 68
100 0
34 0
* Seeds stored for 3 months at 30% at indicated r.h. t One hundred seeds per sample cultured on 10 per cent NaCl-malt agar without sodium hypochlorite pre-treatment. z One hundred seeds per sample cultured on 10 per cent NaCl-malt agar after 1 min in 1 per cent sodium hypochlorite.
In an attempt to reveal the presence of storage fungi in seed lots that were found to be ‘free’ of them by prior plating, yet which yielded them in substantial numbers after storage for 3 months at 30°C and 75 per cent r.h., the following methods were used: (a) Soaking of seeds for as long as 24 hr prior to plating them on 10 per cent NaCl-malt agar. (b) Vigorously washing the seeds in sterile water plus a wetting agent, and incorporation of this washing water into melted agar which was poured
Some Influences
of Storage
Fungi on the Germinability
of Grass Seeds
341
into plates. (c) Comminution of seeds and addition of the resulting slurry to agar which was poured into plates. The results were negative in every case (data not given). DISCUSSION
Seeds of low moisture content stored under conditions unfavourable to the acquisition of additional moisture usually maintain high germinability (EDMOND, 1962 ; GANE, 1947; KEARNS and TOOLE, 1939; TOOLE et al., 1948). In practice, however, it is necessary to know the limits of temperature and r.h. that will insure retention of high germinability. Seeds of the four species studied appear to retain their original germination for at least 12 months at 10°C and 95 per cent r.h. Conversely, maintenance of low r.h. at temperatures up to 35°C resulted in fairly high germination. However, dehumidification of storage areas would probably cost less than refrigeration or air conditioning. Considering the temperature, r.h., and time combinations used in this study, 55 sets of data are available for each species (data not given). Thirty-nine combinations were favourable and five were unfavourable for retention of viability by all foul species. The other eleven combinations provided data by which the relative storability of the seeds used in this study could be differentiated. Mean germinations of the different species, under the eleven ‘stress condi,tions’ were as follows: Poa pratensis, 43 ‘9 per cent; Festuca rubra, 30 - 7 per cent; F. arundinacea, 47 *4 per cent; and Lo&m mult$orum, 81 a6 per cent. Germination of L. multiJIorum at the beginning of the experiment was 7 per cent higher than the average of the other three species. After compensating for this differential, the mean germination of L. multijorum was 34 per cent higher under these ‘stress conditions’ than the mean of the others. A. gfaucus spp. associated with grass seeds evidently play an important part in destroying germinability, but their precise role is difficult to assess because of the lack of fungus-free seeds for comparison. Isolation of these species from a large number of stored seeds was not correlated with a large decrease in germination, since at lower temperatures and r.h., seeds retained high germination in spite of a high incidence of fungi. At high temperature-r.h., germination declined considerably and A. glaucus spp. were usually isolated from many seeds. Although the effects of these fungi could not be separated from internal processes in the seeds, an interaction between them seems probable. Apparently, A. ghcus spp. tended to die out with time in seeds stored at lower levels of temperature and r.h.; however, our failure to culture these fungi from seeds stored up to 12 months did not necessarily prove that they were dead. CHRISTENSENand LOPEZ (1963) state that seeds of barley, wheat, and corn, and probably of other species, are not invaded to any significant degree by storage fungi before harvest. TUITE and CHRISTENSEN(1957) found that less than 5 per cent of the seeds in wheat samples obtained directly from the field and washed or rinsed in 1 per cent sodium hypochlorite prior to plating yielded A. gfaucus spp. However, storage fungi will usually grow from freshly-harvested seeds of wheat and barley when they are cultured on NaCl-malt agar without sodium hypochlorite treatment. We found that, unlike the cereal grains, only field fungi were isolated from many samples of grass seeds when cultured on NaCl-malt agar without surface disinfestation. Nevertheless, A. glaucus spp., particularly A. amstelodami, grew from many
3-U
MARTIN
&I. KULIK
and OKEN L. JUSTICE
grass seeds when cultured on NaCl-malt agar subsequent to storage for three months at 30°C and 75 per cent r.h. The sudden ‘appearance’ of storage fungi horn grass seeds that were ‘free’ of them, prior to storage at 30°C and 75 per cent r.h. may be due to antagonism by field fungi. LVe often observed that few field fungi were isolated when A. glaucus spp, predominated. According to CHRISTENSEX (1957), other investigators have also reported that field fungi normally do not persist for extended times in stored seeds. This leads us to tentatively postulate that (a) storage fungi are present on recently haRested grass seeds, but are kept in a ‘dormant’ state by field fungi, (b) constituents of the seeds may contribute to the repression of storage fungi on recently harvested grass seeds, or (c) interactions may exist between the fungi and the seeds. SCHROEDER (1963) suggested that the interaction between two or more species of fungi (field or storage) in a single rice kernel may affect the prevalence of damaged kernels. This study has emphasized the desirability of using micro-organism-free seeds in investigating the effects of the storage environment on seed germinability. Although CHRISTENSEN(1964) reported that he obtained seeds of corn, wheat, and barley from the field free of internal&borne storage fungi, the results of our research with many samples of grass seeds over a three year period, lead us to believe that it is not likely that grass seeds completely free of storage fungi can be obtained from the field. In addition, any method used to disinfest the seeds such as rinsing in sodium hypochlorite could conceivably alter their metabolic processes. For these reasons, we are attempting to produce a supply of seeds under sterile conditions. Use of microorganism-free grass seeds might very well shed light on our idea of possible antagonism between storage fungi and field fungi on recently harvested grass seeds. wish to thank the following seed frms who kindly supplied us with the grass seeds used in this investigation: Asgrow Seed Company, The Barteldes Seed Company, Cook Seed Company, Hansmeier & Son, Inc., Heritage House Products, Inc., Jacklin Seed Company, Inc., Jenks-White Seed Company, Ed. F. Mangelsdorf and Brother, Inc., Northrup, King and Co., Pacific Cooperative, Robert Pease Seed Company, \Vm. G. Scarlett and Co., and Western Farmers Association. Two of our colleagues, Dr. ARNOLDL. LARSENand 1Mr. MICHAEL F. COMBS assisted us in conducting some of the research. Acknowledgements-We
REFEREXCES CHRISTENSEN, C. M. (1957) Deterioration of stored grains by fungi. Bet. Reu. 23, 108-l 34. CHRISTENSEN, C. M. and LOPEZ, F. L. C. (1963) Pathology of stored seeds, Proc. Iat. Seed Test. Assn. 28, 701-711.
CHRISTENSEN, C. M. (1964) Effect of moisture content and length of storage period upon germination percentage of seeds of corn, wheat, and barley free of storage fungi. Phyfopathology 54, 1464-1466.
EDYOND,J. B. (1962) Studies of storage of vegetable seed under warm-humid conditions. Miss. S!. Univ. Tech. Bail. 50, 24 p. G-WE, R. (1947) The effect of temperature, humidity and atmosphere on the viability of chewing’s fescue grass seed in storage. J. Agr. Sci. Camb. 38, 90-92. HART, J, R., FEINSTEIN,L. and GOLIJMBIC, C. (1959) Oven methods for precise measurements of moisture content of seeds. U.S. Dept. Agr. Mkt. Rrs. Rpl. 304, 16 p. JUSTICE,0. L. (1952) Manual for testing agricultural and vegetable seeds. U.S. Dept. Agr. Handb. 30, 440 p. KEARNS,V. and TOOLE, E. H. (1939) Relation of temperature and moisture content to longevity of Chewing’s fescue seeds. U.S. Dept. Agr. Tech. Bull. 670, 27 p.
Some Influences of Storage Fungi on the Germinability
of Grass Seeds
3-l3
O’BRIES, F. E. 11. (1948) The control of humidity by saturated salt solutions. J. Scien~ Ins~rum. 25, 73-76. \\‘illiams 8; \\‘ilkins. Baltimore, RAPER, K. B. and FENNELL, D. I. (1965) The Gems A+rgilfus. 686 p. SCHROEDER,H. \V. (1963) The relation between storage molds and damage in high-moisture rice in aerated storage. Phytopathology 53, 804808. TOOLE, E. H., TOOLE, \‘. and GORMAN, E. A. (1948) Vegetable-seed storage as affected b) temperature and relative humidity. U.S. Dept. Agric. Tech. Bull. 972, 24 p. TLTTE, J. F. and CHRISTENSEN,C. ill. (1957) Grain storage studies XXIII. Time of invasion of wheat seed by various species of Aspergifltu responsible for deterioration of stored grain, and source of inoculum of these fungi. Phytopathology 47, 265-268. Aloisture content of TUTE, J. F. and CHRISTENSEN,C. M. (1957) G rain storage studies XXIV. wheat seed in relation to invasion of the seed by species of the AspergifIus glaucus group, and effect of invasion upon germination of the seed. Phytopathology 47, 323-327.
Res., 1967,
Vol. 3, pp. 335-343.
Pergamon Press Ltd.
Printed in Great Britain.
Some Influences of Storage Fungi, Temperatures and Relative Humidity on the Germinability of Grass Seeds MARTIN
M. KULIK and OREN
L. JUSTICE
Market Quality Research Division, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705, U.S.A. (First receiued 20 February, 1967, and infinal form 30
June,1967)
Abstract--Seeds of Poa pratensis, Festuca rubra, F. arundinacea, and Lolium multz@um were storedat 10, 20, 30, or 35”C, and at 35, 55, 75, or 95 per cent r.h. for up to 12 months. Samples stored at 30°C and 95 per cent r.h. and 35°C and 75 or 95 per cent r.h. de-creased in germinability after 2 months of storage. At 20°C and either 75 or 95 per cent r.h., seeds decreased more slowly in germinative ability than at the higher temperatures but this decrease was appreciable. Seeds stored at 10°C and at 35, 55, 75, or 95 per cent r.h. germinated well after 12 months. Equilibrium moisture content of the seeds after storage at 35 to 75 per cent r.h. for 2, 4, and 8 months varied little among the four species at each relative humidity level and was not affected by temperature in the range of lO-35°C. Fungi of the AspergiUuv glaucus group could not always be isolated from large numbers of seeds in lots that had declined in germinability. Seeds of all four grasses apparently carried superficially-borne species of the A. glaucus group (primarily A. amstefodami) in a ‘dormant’ atate at harvest. Members of this group were isolated from substantial numbers of seeds that had been stored for 3 months at 30°C and 75 per cent r.h. These fungi were not isolated from seeds stored for the same length of time at 30°C and 35 per cent r.h. ; however, field fungi grew from many of these seeds. INTRODUCTION
STORED seeds are subject to deterioration due to fungi, normal physiological processes in the seeds, insect pests, and rodents. CHRISTENSEN (1957), TUITE and CHRISTENSEN (1957), and CHRISTENSEN and LOPEZ (1963), and others, have shown that certain types of moulds, the so-called storage fungi, can invade and destroy cereals and other seeds when storage conditions become favourable for fungal growth. These fungi, mainly members of the genera As#ergillus and Penicillium, and especially species in the Aspergillus glaucus group,* can grow under relatively dry conditions. Thus, these fungi must be taken into consideration whenever seeds are stored. * Hereafter the expression “A. A. glaucus glaucus group (RAPER and FENNELL, 1965).
spp.” will be used to denote species of the Aspergihs
335
336
kfARTIN
hi. KULIK and OREN L.
JUSTICE
A number of factors, including the growth of suburban areas, have contributed to an increased demand for lawn grasses in the United States, leading to larger quantities of these seeds in market channels and in storage. It is important to know the role which storage fungi play in grass seed deterioration, as well as the effects of storage conditions on seed germinability. iVe undertook to identify the storage fungi associated with certain grass seeds and to determine the influence of fungi, as well as temperature and r.h. on the maintenance of high germination after storage. MATERIALS
AND METHODS
At the beginning of this investigation, a study was undertaken to determine the species and numbers of storage fungi associated with grass seeds. Commercially cleaned seeds of Poa pratensis L., Festuca rubra L., Festuca arundinacea Schreb., and Lolium multzjlorum Lam. were obtained from a number of seed companies. Seeds harvested in 1963 were secured in 1964 after commercial storage for one year. Although the storage histories of these samples were not available, they probably represented a typical cross-section of carry-over grass seed stocks. In addition, samples of seeds produced in 1964 and 1965 were obtained after commercial cleaning, but before storage had started. One hundred seeds from each lot were swirled for 1 min in 1 per cent sodium hypochlorire, rinsed briefly with sterile distilled water, and placed on 10, 15, or 20 per cent NaCl-malt agar. Conidia from developing colonies of fungi were transferred to other Petri dishes for the identification of the isolates. Subsequently, seeds of the four above-mentioned species, intended for use in storage studies, were obtained at harvest time from several growers. The seeds were shipped in cloth bags by surface mail and were kept at 22-25°C and 25-40 per cent r.h. while we cleaned them. Trash and light-weight florets were removed with a miniature seed cleaner. To ascertain whether or not storage fungi were present at the beginning of the experiment, seeds from each cleaned sample were placed on 10 per cent NaCl-malt agar without being rinsed first with sodium hypochlorite. Those samples found to be free of storage fungi, as shown by our plating results, were selected for storage studies. Germination and moisture content determinations followed the procedures of JUSTICE (1952) and HART et al. (1959), respectively. At the time the seed was placed in storage, its germination was as follows: Poa pratensis, 89 per cent; Festuca rubra, 96 per cent; F. arundinacea, 95 per cent; and Lolium multiJorum, 98 per cent. Seeds of each species were placed in separate 150 ml glass jars which were then covered with fine cloth gauze to avoid mixing. The jars with the seeds were put into glass desiccators containing saturated salt solutions to produce r.h. of 35, 55, 75, or 95 per cent (O’BRIEN, 1948). The desiccators were placed in incubators held at 10, 20, 30, or 35°C. In this manner, sixteen combinations of r.h. and temperature were maintained. Subsamples were withdrawn after 2, 4, 8, or 12 months. To detect storage fungi, seeds were swirled for 1 min in 1 per cent sodium hypochlorite, followed by a brief rinse with sterile, distilled water, and were then placed on 10 per cent NaCl-malt agar. The plates containing the seeds were incubated at 22-25X. Germination percentages were based on 200 seeds per subsample. Moisture contents (wet weight
Some Influences
of Storage
Fungi on the Germinability
337
of Grass Seeds
basis) were determined at each withdrawal date, except that no moisture determinations were made after 8 months. Two 2-g portions of seeds from each subsample were dried at 130°C for 1 hr in a hot air oven. RESULTS
Fungi associated with grass seedr
A large number of samples of grass seeds from the 1963 carry-over stocks as well as seeds from 1964 and 1965 that had not been stored, were assayed for storage fungi. The results in Table 1 for seeds of Poa pratensis are typical of those obtained from many samples of seeds of Festuca rubra, F. arundinacea, and Lolium mult$orum. The principal storage fungi encountered were A. glaucus spp., with A. amstelodami (Mang.) Thorn and Church most commonly isolated; some other species of Aspergillus were isolated but only in trace amounts (Table 1).
TABLE ON
10
1. per
NUMBER Cent
OF Pea
SODIUM
Year of seed harvestt 1963 1963 1963 1963 1963 1964 1964 1964 1965 1965 1965
pratensis
SEEDS
CHLORIDE-MALT
FROX
WHICH
AGAR
WITHOUT
FUSGI
WERE
SODIC’M
ISOLATED
WHEN
HYPOCHLORITE
Aspergillus glaucus spp.
Penicillium
species
Field fungi:
73 0 0 0 3 0 0 41 0 2 0
3-l 2 7 0 2 0 0 0
24 100 100 100 100 100 100 13 100 100 100
1 1 1
C~‘LTC~RED
TREATMENT*
. * One hundred seeds per sample. 7 Seeds from 1963 were stored commercially for one year prior to receipt; the 1964 and 1965 samples were obtained before storage had occurred. $ Principally Alternaria tenuis auct. sensu Wiltshire and Cladosporium spp. In addition, a few samples contained trace amounts of Aspergillus candidus Link, A. niger V. Tiegham, and A. och?accrrs Wilhelm.
Apparently, the grass seeds we plated were either free of storage fungi or only lightly contaminated; however, an occasional sample was heavily infested. Conversely, field fungi, such as Cladosporium herbarum (Pers.) Link ex Fr. and Alternaria tenuis auct. sensu Wiltshire, were often present in very large numbers; they were sometimes isolated from every seed in a subsample. Surface disinfestation with sodium hypochlorite prior to plating indicated that, in most cases, establishment of field fungi was evidently limited to the seed surface. Hyperosmophilic species, such as Aspergillus halophilicus Christensen, Papavizas, and Benjamin and A. restrictus Smith, were ‘not isolated from grass seeds cultured on 15 or 20 per cent NaCl-malt agar. E
338
~~ARTIN hf. KULIK
Comparison isolated
of seed germination
and OREN L. JUSTICE
with the number of see& from
which A. glaucus spP. were
(Note: The data presented in Table 2 represent an average of the data obtained from the four species of grasses. The individual results were averaged because the performance of the seeds of each grass was quite similar.) A. Seedr storedfor 2 months. No apparent correlation existed between germination and the number of seeds from which A. glaucus spp. were isolated. Seeds of the four species taken from samples stored at 12 of 16 temperature-r.h. conditions germinated no lower than 90 per cent, yet the number of seeds per sample from which A. glauczu spp. were isolated varied from 1 to 99 per cent (Table 2). To further illustrate this disparity between number of seeds yielding these fungi and seed viability, these species were isoIated from 84 per cent of seeds taken from samples stored at 35°C and 75 per cent r.h. that germinated 16 per cent. However, A. glaucus spp. were obtained from 96 per cent of seeds from samples stored at 30°C and 95 per cent r.h. that germinated 53 per cent. 2. PERCENTAGEOF GRASS SEEDS FROM WHICHAspergillus glaucw spp. WERE ISOLATED AND per cent GERMINATION AFIZR STORAGE FOR ‘2-12 months UNDER 16 TEMPERATURE-RELATWE
TABLE
HUMIDITY COMBINATIONS*
Relative humidity 35%
Months in storage 2
Storage temp. (“C)
0I
5 1 47 84
1 5 29 20
91 92 89 85
94 94 97 93
1 2 9 2
90 94 93 88
0 1 1 1
16 3 27 48
10
10 20 30 35
O’ Ge?m.
92 91 77 16
7 99 96 93
92 90 53 1
2 50 100
88 88 16
3 100 95
92 83 0
94 95 91 49
0 95
93 64
7 45
94 4
90 94 73 19
1 66
92 27
1
92
93 94 94 94
6 2 8 31
5 3 50 77
91 88 92 92
1 2 26 7 1 5 1
Freqcency A. glaucus SPP*
SPP.
y(, Germ.
0
Freqzenc y A. glaucus
91 92 93 91
10 20 30 35
10
0’
01
SPP*
SPP.
20 30 35
FreqEenc y A. glaucus
95%
“/b Germ.
“/;, Germ.
20 30 35
12
% Frequency A. glatuus
75%
55%
* Each value represents a mean for the four species: Poa pratcnsk, Fcstuca rubra, F’. arundinacea, and Lo&urn muft;POrum. One hundred seeds per sample were cultured on 10 per cent NaCl-malt agar after sodium hypochlorite treatment, and 200 seeds per sample were used to determine germination.
Some Influences of Storage Fungi on the Germinability of Grass Seeds
339
After 2 months, a decline in germination was noted for seed stored at combinations of 30 and 35°C and 75 and 95 per cent r.h. (Table 2). Samples of very low germinability were omitted from this invesbgation after the evaluation at 2 months was completed, and also at 4 and 8 months. B. Seeds storedfor 4 months. Germinability remained high at 10 of the 14 Icvels of temperature and r.h., i.e. 88 per cent or better. The trend toward loss in germination at higher levels of temperature and r,h. continued with a slight loss in germination in seeds stored at 35°C and 55 per cent r.h. (down from 9 1 to 85 per cent after 2 months). Great variation was observed in the number of seeds from which A. gluucus spp. were isolated. This ranged from 1 to 100 per cent with a germination level no lower than 83 per cent in 12 out of 14 samples. C. See& stored for 8 months. Seeds stored under 9 of the 12 conditions germinated 91 per cent or higher. The number of high germinating seeds from which A. glaucus spp. were isolated dropped considerably, i.e. the range was now 0 to 26 per cent compared to 1 to 77 per cent at 4 months. Germination continued to decrease at the higher temperatures and r.h., dropping 42 per cent at 35°C and 55 per cent r.h., 27 per cent at 20°C and 75 per cent r.h., and 95 per cent at 20°C and 95 per cent r.h. (Table 2). D. Seeds stored for 12 months. Almost no A. glaucus spp. were isolated from seeds stored at 10 of the 11 conditions and germination of seeds from 8 of the 11 conditions was no lower than 88 per cent. Germination of lots stored at the higher temperatures and r.h. continued to decrease. For example, decreases between the 8-month and 12-month storage periods were 20 per cent at 30°C and 55 per cent r.h., 61 per cent at 35°C and 55 per cent r.h., and 58 per cent at 20°C and 75 per cent r.h. (Table 2). Curiously, at 20°C and 75 per cent r.h. the percentage frequency of A. glaucus spp. had decreased from 95 to 66 within the 4-month period, although the germination had dropped from 64 to 27 per cent. Moisture
content of seeds at d$erent
temperature-relative
humidity levels
Moisture contents of seeds of the four species studied were determined on a wet weight basis after 2, 4, and 8 months of storage (Table 3). The raw data (not presented) indicated that temperature over a range of lo-35°C had little effect on the moisture content of seeds stored at 35-75 per cent r.h. Moisture contents of seeds of the four grasses used in this investigation varied no more than I per cent among species at a given r.h., except for F. yubra at 95 per cent r.h. (Table 3). These moisture contents remained unchanged during the 8-month storage period. Isolation of A. glaucus plating methods
spp.ji'om seeds apparently ‘free’ of storagejilngi
according to standard
A. glaucus spp. were subsequently isolated from seeds in certain samples that previously had been found ‘free’ of storage fungi by the agar plating procedure. To gain some insight into the cause of this phenomenon, grass seed lots were assayed in 1964 and 1965 for the presence of storage fungi and those found to be ‘free’ of them were stored at 35 or 75 per cent r.h. at 30°C. After 3 months of storage, these seeds were assayed in 10 per cent NaCl-malt agar. The results for each year are summarized as follows: (1) A. gluucus spp, were not isolated from seeds taken from
&fARTIN xi. I(ULIK and OREN
340
TABLE
L.JUSTICE
3. AVERAGE per cent MOISTURE (WET WEIGHT BASIS)OF GRASS SEEDS STORED AT SIXTEEN TEUPERATURE-RELATIVE HUMIDITY COMBINATIONS*
Moisture content/relative humidity Species of grass Poa prateffsis Festka rubra Festuca arundinacea Ldium muftfJ?orfmf
350,;
55:,;
75?,
95”0
8.4
IO-3 10.9 11.0 11.3
13.5 14.1 13.8 14.2
17.3 20.3 18.3 18.1
8.8 9-l
9.1
* Data represent the combined average moisture contents of samples stored at 10, 20, 30, or 35°C and measured at intervals of 2, 4, or 8 months.
samples stored for 3 months at 30°C and 35 per cent r.h., but field fungi were isolated from every seed in almost all samples (Table 4). (2) All seeds from samples stored at 30°C and 75 per cent r.h. for 3 months yielded A. glaucus spp. but no field fungi (Table 4). TABLE 4. INFLUENCE OF STORAGE ENVIROSMENT ON THE NUMBER OF GRASS SEEDS GROWN IN 1964 AND 1965 FROM WHICH Aspergillw glaucuJ spp. AND FIELD FUNGI WERE ISOLATED A. glaucus spp.
Species of grass Poa pratensis
Festuca rubra
No. of samples averaged 5
3
Festuca ar ffndinacea
2
Lolifim JnJfLtJgforuJJl
5
Tb r.h. of storage*
Total seedborne populationt 0
35 75
100
35 75
100
35 75
100
35 75
100
0
0
0
Internallyborne populationS
Field fungi Total seedborne populationf
Internallyborne population:
0 33
98 0
11 0
0 37
100 0
26 0
0 62
100 0
33 0
0 68
100 0
34 0
* Seeds stored for 3 months at 30% at indicated r.h. t One hundred seeds per sample cultured on 10 per cent NaCl-malt agar without sodium hypochlorite pre-treatment. z One hundred seeds per sample cultured on 10 per cent NaCl-malt agar after 1 min in 1 per cent sodium hypochlorite.
In an attempt to reveal the presence of storage fungi in seed lots that were found to be ‘free’ of them by prior plating, yet which yielded them in substantial numbers after storage for 3 months at 30°C and 75 per cent r.h., the following methods were used: (a) Soaking of seeds for as long as 24 hr prior to plating them on 10 per cent NaCl-malt agar. (b) Vigorously washing the seeds in sterile water plus a wetting agent, and incorporation of this washing water into melted agar which was poured
Some Influences
of Storage
Fungi on the Germinability
of Grass Seeds
341
into plates. (c) Comminution of seeds and addition of the resulting slurry to agar which was poured into plates. The results were negative in every case (data not given). DISCUSSION
Seeds of low moisture content stored under conditions unfavourable to the acquisition of additional moisture usually maintain high germinability (EDMOND, 1962 ; GANE, 1947; KEARNS and TOOLE, 1939; TOOLE et al., 1948). In practice, however, it is necessary to know the limits of temperature and r.h. that will insure retention of high germinability. Seeds of the four species studied appear to retain their original germination for at least 12 months at 10°C and 95 per cent r.h. Conversely, maintenance of low r.h. at temperatures up to 35°C resulted in fairly high germination. However, dehumidification of storage areas would probably cost less than refrigeration or air conditioning. Considering the temperature, r.h., and time combinations used in this study, 55 sets of data are available for each species (data not given). Thirty-nine combinations were favourable and five were unfavourable for retention of viability by all foul species. The other eleven combinations provided data by which the relative storability of the seeds used in this study could be differentiated. Mean germinations of the different species, under the eleven ‘stress condi,tions’ were as follows: Poa pratensis, 43 ‘9 per cent; Festuca rubra, 30 - 7 per cent; F. arundinacea, 47 *4 per cent; and Lo&m mult$orum, 81 a6 per cent. Germination of L. multiJIorum at the beginning of the experiment was 7 per cent higher than the average of the other three species. After compensating for this differential, the mean germination of L. multijorum was 34 per cent higher under these ‘stress conditions’ than the mean of the others. A. gfaucus spp. associated with grass seeds evidently play an important part in destroying germinability, but their precise role is difficult to assess because of the lack of fungus-free seeds for comparison. Isolation of these species from a large number of stored seeds was not correlated with a large decrease in germination, since at lower temperatures and r.h., seeds retained high germination in spite of a high incidence of fungi. At high temperature-r.h., germination declined considerably and A. glaucus spp. were usually isolated from many seeds. Although the effects of these fungi could not be separated from internal processes in the seeds, an interaction between them seems probable. Apparently, A. ghcus spp. tended to die out with time in seeds stored at lower levels of temperature and r.h.; however, our failure to culture these fungi from seeds stored up to 12 months did not necessarily prove that they were dead. CHRISTENSENand LOPEZ (1963) state that seeds of barley, wheat, and corn, and probably of other species, are not invaded to any significant degree by storage fungi before harvest. TUITE and CHRISTENSEN(1957) found that less than 5 per cent of the seeds in wheat samples obtained directly from the field and washed or rinsed in 1 per cent sodium hypochlorite prior to plating yielded A. gfaucus spp. However, storage fungi will usually grow from freshly-harvested seeds of wheat and barley when they are cultured on NaCl-malt agar without sodium hypochlorite treatment. We found that, unlike the cereal grains, only field fungi were isolated from many samples of grass seeds when cultured on NaCl-malt agar without surface disinfestation. Nevertheless, A. glaucus spp., particularly A. amstelodami, grew from many
3-U
MARTIN
&I. KULIK
and OKEN L. JUSTICE
grass seeds when cultured on NaCl-malt agar subsequent to storage for three months at 30°C and 75 per cent r.h. The sudden ‘appearance’ of storage fungi horn grass seeds that were ‘free’ of them, prior to storage at 30°C and 75 per cent r.h. may be due to antagonism by field fungi. LVe often observed that few field fungi were isolated when A. glaucus spp, predominated. According to CHRISTENSEX (1957), other investigators have also reported that field fungi normally do not persist for extended times in stored seeds. This leads us to tentatively postulate that (a) storage fungi are present on recently haRested grass seeds, but are kept in a ‘dormant’ state by field fungi, (b) constituents of the seeds may contribute to the repression of storage fungi on recently harvested grass seeds, or (c) interactions may exist between the fungi and the seeds. SCHROEDER (1963) suggested that the interaction between two or more species of fungi (field or storage) in a single rice kernel may affect the prevalence of damaged kernels. This study has emphasized the desirability of using micro-organism-free seeds in investigating the effects of the storage environment on seed germinability. Although CHRISTENSEN(1964) reported that he obtained seeds of corn, wheat, and barley from the field free of internal&borne storage fungi, the results of our research with many samples of grass seeds over a three year period, lead us to believe that it is not likely that grass seeds completely free of storage fungi can be obtained from the field. In addition, any method used to disinfest the seeds such as rinsing in sodium hypochlorite could conceivably alter their metabolic processes. For these reasons, we are attempting to produce a supply of seeds under sterile conditions. Use of microorganism-free grass seeds might very well shed light on our idea of possible antagonism between storage fungi and field fungi on recently harvested grass seeds. wish to thank the following seed frms who kindly supplied us with the grass seeds used in this investigation: Asgrow Seed Company, The Barteldes Seed Company, Cook Seed Company, Hansmeier & Son, Inc., Heritage House Products, Inc., Jacklin Seed Company, Inc., Jenks-White Seed Company, Ed. F. Mangelsdorf and Brother, Inc., Northrup, King and Co., Pacific Cooperative, Robert Pease Seed Company, \Vm. G. Scarlett and Co., and Western Farmers Association. Two of our colleagues, Dr. ARNOLDL. LARSENand 1Mr. MICHAEL F. COMBS assisted us in conducting some of the research. Acknowledgements-We
REFEREXCES CHRISTENSEN, C. M. (1957) Deterioration of stored grains by fungi. Bet. Reu. 23, 108-l 34. CHRISTENSEN, C. M. and LOPEZ, F. L. C. (1963) Pathology of stored seeds, Proc. Iat. Seed Test. Assn. 28, 701-711.
CHRISTENSEN, C. M. (1964) Effect of moisture content and length of storage period upon germination percentage of seeds of corn, wheat, and barley free of storage fungi. Phyfopathology 54, 1464-1466.
EDYOND,J. B. (1962) Studies of storage of vegetable seed under warm-humid conditions. Miss. S!. Univ. Tech. Bail. 50, 24 p. G-WE, R. (1947) The effect of temperature, humidity and atmosphere on the viability of chewing’s fescue grass seed in storage. J. Agr. Sci. Camb. 38, 90-92. HART, J, R., FEINSTEIN,L. and GOLIJMBIC, C. (1959) Oven methods for precise measurements of moisture content of seeds. U.S. Dept. Agr. Mkt. Rrs. Rpl. 304, 16 p. JUSTICE,0. L. (1952) Manual for testing agricultural and vegetable seeds. U.S. Dept. Agr. Handb. 30, 440 p. KEARNS,V. and TOOLE, E. H. (1939) Relation of temperature and moisture content to longevity of Chewing’s fescue seeds. U.S. Dept. Agr. Tech. Bull. 670, 27 p.
Some Influences of Storage Fungi on the Germinability
of Grass Seeds
3-l3
O’BRIES, F. E. 11. (1948) The control of humidity by saturated salt solutions. J. Scien~ Ins~rum. 25, 73-76. \\‘illiams 8; \\‘ilkins. Baltimore, RAPER, K. B. and FENNELL, D. I. (1965) The Gems A+rgilfus. 686 p. SCHROEDER,H. \V. (1963) The relation between storage molds and damage in high-moisture rice in aerated storage. Phytopathology 53, 804808. TOOLE, E. H., TOOLE, \‘. and GORMAN, E. A. (1948) Vegetable-seed storage as affected b) temperature and relative humidity. U.S. Dept. Agric. Tech. Bull. 972, 24 p. TLTTE, J. F. and CHRISTENSEN,C. ill. (1957) Grain storage studies XXIII. Time of invasion of wheat seed by various species of Aspergifltu responsible for deterioration of stored grain, and source of inoculum of these fungi. Phytopathology 47, 265-268. Aloisture content of TUTE, J. F. and CHRISTENSEN,C. M. (1957) G rain storage studies XXIV. wheat seed in relation to invasion of the seed by species of the AspergifIus glaucus group, and effect of invasion upon germination of the seed. Phytopathology 47, 323-327.