Effects of Broiler Litter Volatiles and Ammonia on Fungal Spore Germination

Effects of Broiler Litter Volatiles and Ammonia on Fungal Spore Germination c. w. BACON Toxicology and Biological Constituents Research Unit, R. B. Russell Agricultural Research Center, USDA, ARS, Athens, Georgia 30613 (Received for publication June 17, 1985)

1986 Poultry Science 65:710-716 INTRODUCTION

Although fungal growth has been demonstrated to occur in broiler litter (Bacon and Burdick, 1977), most fungi exist in the litter as spores (Dennis and Gee, 1973; Lovett et al, 1971; Halbrook et al, 1951). The ability of spores to germinate and subsequently grow on litter substrate where toxic compounds are often produced depends on various environmental factors. Although the precise environmental factors were not clearly established, Bacon and Burdick (1977) discussed these factors relative to growth and presented data suggesting that the growth of fungi in litter was associated with specific houses and was related to management practices. One of the major environmental controls of spore germination in soil is the phenomenon of soil fungistasis (Lockwood, 1977). As a consequence of fungistasis, a spore remains dormant in the soil without germination, although there are adequate conditions for it to germinate. This phenomenon also represses fungal growth. Many fungistatic substances are volatile, however, not many have been characterized chemically (Balis andKouyeas, 1968). Recently, ammonia was identified as a volatile fungistatic substance from alkaline soils (Ko et al, 1974; Schippers and Pruis, 1978) and its effects on the germination of a number of soil fungi reported (Schippers et al, 1982). This paper examines the role of spore fungistasis in broiler litter and the effects of

ammonia on the germination of fungal spores in broiler litter, in particular the spores of several toxic fungi. MATERIALS AND METHODS Litter Samples and Test Fungi. Broiler house litter used in the study was collected at the end of a 7-week rearing period from two different broiler flocks. The litter from the first flock was fresh litter initially, while that from the second flock was built-up litter that had one prior flock. Litter used for producing the fungistatic compound was air-dried for 2 days and stored until use at —20 C in a closed container. The fungi used in this study included several species isolated from broiler houses and reported as growing in litter (Bacon and Burdick, 1977): Aspergillus clavatus, A. flavus, A. fumigatus, A. ochraceus, Cladosporium cladosporioides, Fusariumpoa, Oidiodendron fuscum, Penicillium cyclopium, P. patulum, Scopulariopsis brevicaulis, Trichoderma viride, and Trichothecium roseum. All fungi were maintained on Difco Czapek Agar with 1% Difco yeast extract added (CZY medium). Spores used for germination studies were harvested from 1-month-old cultures grown on CZY medium. Fungistatic Assay of Volatile Inhibitor. The apparatus used to determine the effects of volatile inhibitors in litter was modified from

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ABSTRACT The effects of broiler litter volatiles on spore germination of 12 fungal species were determined. The volatiles inhibited spore germination of all species except Scopulariopsis brevicaulis. The inhibitory effect was fungistatic. The inhibitory volatile substances in litter were higher in built-up litter than in freshly used litter and were soluble in an acid solution. Ammonia was identified by infrared analysis as a fungal inhibitory substance in litter. The effect of various concentrations of ammonia produced from ammonium chloride (NH4C1) was studied, and it was determined that concentrations of ammonia below 150 jug/g dry weight of litter were fungistatic for most species. At higher concentrations, ammonia was fungitoxic, with the exception of S. brevicaulis whose spore germination was not inhibited at this high concentration. Fungitoxic levels of ammonia were not found in the litter samples, but broiler house conditions necessary to produce fungitoxic levels are discussed. (Key words: litter volatile, spore germination inhibition, fungistasis, litter ammonia, toxic fungi)

INHIBITORY EFFECTS OF LITTER AMMONIA

Spores (10 4 /ml) of a test fungus were inoculated on sterile dialysis membranes placed on the surface of the CZY medium. The lid was removed and each dish placed into the incubation chamber. Spores used as a control were germinated similarly to air passed through the purifying train described above, except litter was omitted. Control spores of all fungi completed germination within a 24-hr period at 20 C, therefore germination was assessed within 24 hr of exposure to the litter column effluent. The dialysis tubing was removed from the CZY medium surface, placed on a microscope slide, and approximately 200 spores were observed for germination. Spores were considered germinated when the germ tube length equaled half the length of the spore. Unused litter, treated in the identical manner as described above, was used as a control. The data were analyzed using analysis of variance and Duncan's multiple range test. The use of the term significance in this paper refers to P<.05. Identification of Fungistatic Compound. The alkaline nature of the fungistatic substance in litter was established by comparing conidial germination in litter effluent treated either with or without an acid trap of 100 ml of .05 M boric acid (H3BO3). The fungistatic substance was collected for chemical identification by passing the air effluent from the litter-filled column described above through an acid trap of 100 ml of .05 M hydrochloric acid (HCl) for a 12-hr period. The HCl solution was evaporated to dryness at 90 C, the residue mixed into a potassium bromide (KBr) pellet, and analyzed by infrared spectroscopy (Fortran IR spectrometer, Model FX-6160).

The amount of ammonia in the traps was determined by direct nesslerization on aliquots of the acid solution neutralized with 2.5 N sodium hydroxide (NaOH) (Thomas and Chamberlin, 1974). Ammonia in the broiler litter was determined on a filtrate obtained by extracting 10 g of freshly collected litter with 100 ml of 2 M potassium chloride (KC1) following the procedure of Keeney and Nelson (1982); it was quantitated by direct nesslerization on an aliquot of the filtrate using the Conway microdiffusion procedure of Bremner (1965). Moisture, pH, and total fungal count were also determined on freshly collected litter following the procedures described earlier (Bacon and Burdick, 1977). The effect of different ammonia concentrations on spore germination was determined in sealed Conway vessels as described by Schippers et al. (1982). The following procedure was used to produce the various concentrations in the atmosphere of the vessel. Air-dried, fresh pine shavings were ground through a 4-mm sieve of a Wiley mill. Various concentrations of aqueous NH4C1 solutions were added to weighed amounts of the shavings that were placed in the outer ring of the Conway vessel. The membrane containing test spores was placed over nutrient agar in the center ring of the vessel, the vessel sealed, and ammonia was liberated by injecting .05 M NaOH through the stopper to saturate the shavings. Control treatments for each ammonia concentration consisted of identical amounts of shavings containing identical volumes of water and NaOH. The Conway vessels were incubated for 24 hr at 20 C. When it was desired to trap the ammonia in the Conway vessels, 2 ml of .05 M H 3 B 0 3 was placed in the center well, and the dialysis tubing containing the spores was attached to the lid of the vessel. The dialysis tubing in this case was coated with a thin film of CZY medium.

RESULTS

Built-up litter contained fewer fungal propagules and was significantly higher in moisture, pH, and concentration of ammonia than new litter (Table 1). The concentration of ammonia was approximately 7.5 times higher than that in new litter. Not only was the new litter higher in total numbers of fungi, but this litter also contained 39 species of fungi with several species occurring in equal relative

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Romine and Baker (1972). Air was used to remove volatiles from the litter at a rate of 2 ml/min. The air was purified by passing it through a charcoal trap, double-distilled sterile water, 50% sulfuric acid (H2SO4), doubledistilled sterile water, and drierite. The purified air was then passed through a glass column (125 X 9.5 cm i.d.) of litter ( l k g ) that was moistened with double-distilled sterile water to approximately 50% moisture content. The effluent from the litter-filled column was passed to a manifold and from it to eight separate spore incubation Petri dish chambers (15 X 2 cm) of the Romine and Baker (1972) apparatus. Each spore incubation Petri dish chamber contained four smaller plastic Petri dishes (6 x 1.5 cm) of CZY medium upon which germination tests were conducted.

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TABLE 1. Characteristics of the two litter types used for fungistatic factor Litter type'

New litter Built-up litter

pH

7.1 + . l a 8.9 ± . l b

Moisture

Total fungi

NH4-N

(%)

(counts/g wet wt)

(Mg/g dry wt)

29.3 ± 1.2a 33.2 ± . 9 b

4.3 X 10 6 ± 1.0a 2.3 X 10 s + 2 . 1 b

18.9 ± .9 a 134.6 ± . 7 b

ab ' Means (± SE) in a column with different superscripts are significantly different (P<.05).

density (the number of one species isolated relative to the total number of isolated species). In contrast, the total number of fungal species in built-up litter was 15, which was mainly represented by S. brevicaulis (45% relative density) and T. roseum (24% relative density). Spores of the fungi isolated from litter were tested for their ability to germinate in an atmosphere of broiler litter. Spore germination of most fungi was inhibited significantly under atmospheres from new and built-up litter (Table 2). Inhibition of spore germination never occurred under an atmosphere from unused litter controls. Germination was less for spores treated with gaseous effluent from built-up litter than that achieved with litter used only once. The germination of S. brevicaulis was not significantly inhibited by either litter type

(Table 2). Inhibited spores germinated when they were removed from the broiler litter atmosphere and placed into fresh air. Inhibition of spore germination was prevented when the effluent air was passed through an acid trap before it entered the spore germination chamber (Table 3). The volatile substance was trapped in the acid as a chloride salt. The salt was subjected to infrared spectrometry, and the major absorption maxima of ammonia chloride in KBr were obtained (max (KC1): 1404, 3047, 3140 cm" 1 ) that were identical to authentic NH4C1 and similar to published spectra (Anderson and Woodall, 1953). This result established that the volatile compound trapped was ammonia. There was no spectral evidence for the presence of other volatile compounds in the litter effluent trapped

TABLE 2. Effect of volatile fungistatic factor from two types of boiler litters on fungal spore germination % Germination 1 Species

Controls

litter

Built-up litter

Aspergillus clavatus A. flavus A. fumigatus A. ochraceus Cladosporium cladosporioides Fusarium poa Oidiodendron fuscum Penicillium cyclopium P. patulum Scopulariopsis brevicaulis Trichoderma viride Tricho tbecium roseum

9Sa 98a 99a 96a 94a 98a 81a 93a 91a 99a 96* 88a

31b 28b 25b 20b 50b 41b 57b 47b 39b 99a 61b 57b

5C 2C 3C 0C 19c 18c 19c 5C 6C 97a 22C 29c

New

' ' Means in a row within different superscripts are significantly different (P<.05). 1

Inhibited spores germinated when placed into fresh air.

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1 Sampling and analysis performed at the end of the rearing period, 7 weeks; mean ± SE of 10 separate analyses of a composite sample that consisted of 40 individual fresh samples, randomly collected throughout the broiler houses.

INHIBITORY EFFECTS OF LITTER AMMONIA

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TABLE 3. Effect of acid trap on the activity of a volatile fungistatic factor from built-up broiler litter on fungal spore germinations % Germination 1

Controls

Aspergillus clavatus A. flavus A. fumigatus A. ochraceus Cladosporium cladosporioides Fusarium poa Oidiodendron fuscus Penicillium cyclopium P. natulum Scopulariopsis brevicaulis Trichoderma viride Trichotbecium roseum

94a 97a 98a 99a 97a 96a 84a 97a 98a 79a 97a 90a

Air passed through litter and acid trap

2b

90a 84c 81a 75C 95a 92a 79a 80a 83c 83a 93a 95a

3b 5b 2b 22b 17b 25b 9b 8b 94b 17b 21b

' ' Means in a row within different superscripts are significantly different (P<.05). 1

Values are means of four replicates; inhibited spores germinated when placed into fresh air.

in the acid solution. The effect of ammonia, produced from different concentrations of NH4CI in Conway dishes on spore germination, established that germination was inhibited by ammonia, and that the degree of inhibition was

species and concentration dependent (Table 4). Of the species tested, A. parasiticus and A. ochraceus were the most sensitive to all ammonia concentrations. Inhibition of spore germination of these two fungi was complete,

TABLE 4. Spore germination after a 24-hr exposure period to various concentrations of ammonia produced from ammonium chloride (NHACl) Conidial germination, %' MgNH 4 - -N/g shavings Species

.1

1.0

5.0

10.0

50

100

Aspergillus flavus3 A. fumigatus3 A. ochraceus3 A. parasiticus3 Cladosporium cladosporioides Fusarium poa Oidiodendron fuscum Penicillium cyclopium3 P. patulum Scopulariopsis brevicaulis Trichoderma viride Trichotehcium roseum

37a 45a 16a 15a 85a 83" 87a 76a 44a 81a 87a 96a

4b 47a 2a 5a 79a 81a 77a 65a 7b 91a 76a 40b

3b 20b la 0 43b 25b 81a 6b 3b 94a 30b 22b

0 10b 0 0 14c 36b 40b 3b 2b 99a

0 0 0 0 10c 10b

0 0 0 0 0 0 10c 2b 3b 97a 7C 0

14bc

3C

2obc 5b lb 98a 5C lc

NH 4 - N and Boric acid2 98c 93C 80b 72b 94a 99a 93a 94c 90c 90a 89a 91a

' ' Means in a row within different superscripts are significantly different (P<.05). 1

All values are means of four replicates.

2

Boric acid was used as a trapping agent in the Conway chamber only with the 50-jug NH4—N level.

3 Spores of these species did not germinate at concentrations of NH„— N at 150 Mg/g and above when placed into fresh air.

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Species

Air passed through litter only

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DISCUSSION Spore germination of 11 out of 12 species of fungi was inhibited by a volatile substance from broiler litter. The volatile substance has alkaline properties, because there is loss in inhibition of spore germination when the litter effluent is first passed through an acid trap before being delivered to the spore germination chamber. It was determined that the trapped substance was

ammonia. When NH4C1 was added to unused litter, only ammonia was present in the atmosphere, and spore germination was inhibited. Further, in an atmosphere of ammonia, spores germinated when boric acid was placed in the center well of the Conway cell. It is concluded that ammonia is a spore fungistatic substance in broiler litter. The involvement of ammonia as a volatile fungistatic substance in soil was first reported by Ko et al. (1974), but the role of it, as well as other volatile substances, as a widespread inhibitor in all soil types was considered doubtful (Lockwood, 1977) because the production of ammonia requires a unique soil type, namely alkaline forest soil rich in organic matter. Nevertheless, the results of others indicated that ammonia is a common volatile spore inhibitor (Paulica et al, 1978; Schippers and Palm, 1973; and Ko et al, 1974). My study adds support and indicates an ammonia-generating system in an isolated, but real, situation. Broiler litter is rich in organic matter because of its excreta content. The excreta serves as a biological source of ammonia through the direct and indirect decomposition of uric acid into ammonia as has been demonstrated with strains of Corynebacterium and five other species of bacteria (Schefferle, 1965). Therefore, the origin of ammonia is biological. The level of ammonia generated during the 12-hr period from the two litter types is probably high, because the biological activity of the

TABLE 5. The occurrence and assay of a fungistatic factor in broiler litter from different farms % Germination' Litter type 2

F; Farm

Aspergillus ocbraceus

New New New Built-up Built-up Built-up

1 2 3 4 5 6

l\a 7* 25 a 5a 3a 2a

Scopulariopsis brevicaulis

98b 96b 97b 98° 99b 96 b

NH4-N (Mg/g dry wt) 9.3 C 5.3 C 8.4C 139.4 d 132.3 d 121.2 d

a ' b Means in a row within percent germination category with different superscripts are significantly different (P<.05). c ' d Means in a column within NH — N category with different superscripts are significantly different (P<.05). 4 1

Mean value of four replicates; all spores of A. ocbraceus germinated when placed into fresh air.

2

Litter collected at the end of rearing period, 7 weeks; built-up litter used for two broiler flocks.

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or almost complete, except at the lowest concentration where about 85% inhibition was observed. Litter from six different broiler farms was assayed for the presence of a volatile inhibitor. Only spores of A. ocbraceus and 5. brevicaulis were used to test for a volatile substance. Results from these six locations (three built-up litter and three used litter once) indicated that a volatile spore inhibitor was present and that spores of S. brevicaulis were not inhibited (Table 5). The amount of ammonia contained in litter from each farm was determined within 12 hr of collecting the sample, and the ammonia content during this period was 9 to 27 times more in built-up litter than in litter used only once for the rearing of birds. As indicated above, A. ocbraceus was extremely sensitive to ammonia (Table 4), therefore, the small amount of ammonia present in new litter was sufficient to produce statistically the same inhibition as that produced on built-up litter (Table 5).

INHIBITORY EFFECTS OF LITTER AMMONIA

sphere required to control spore germination is low and would pose no problems to broilers. Spores used in this study were those that either required, or did not require, outside nutrients for germination, and the inhibitory nature of ammonia did not distinguish either spore type. For example, S. brevicaulis required nutrients, and germination was completed as long as this requirement was met in spite of the persence of ammonia. The complete absence of an effect of broiler litter volatiles on the germination of spores of S. brevicaulis might explain the high density of its propagules in litter (Halbrook et al, 1951; Forgacs et al, 1962; Lovett et al, 1971) and its high growth rate in litter (Bacon and Burdick, 1977). The ineffectiveness of ammonia on the germination and growth of some fungi has been reported earlier (Ko et al, 1974; Schippers et al, 1982). The growth of S. brevicaulis and other fungi have been shown to be stimulated by ammonia, but unlike other fungi, S. brevicaulis is not sensitive to pH changes and indeed at alkaline pH, as occurs in the litter, the uptake and utilization of ammonia are stimulated (Morton and MacMillan, 1954). The effects of broiler litter volatiles on those fungi that have already germinated before inhibitory levels of ammonia were produced, and on their ability to produce mycotoxins, are unknown. This aspect of the problem should be examined to define the role, if any, of litter fungi in poultry mycotoxicoses and to distinguish their involvement from toxigenic field and storage fungi known to impair poultry performance. ACKNOWLEDGMENTS I thank R. M. Bennett for technical assistance and D. S. Himmelsbach, Plant Structure and Composition Research Unit, USDA— ARS, for infrared analysis. The litter materials used in this study were obtained through the courtesy of G. Hunter, whose help is greatly appreciated. REFERENCES Anderson, D. H., and N. B. Woodall, 1953. Infrared identification of materials in the fractional milligram range. Anal. Chem. 25:1906-1907. Anderson, D. P., C. W. Beard, and R. P. Hansen, 1964. The adverse effects of ammonia on chickens including resistance to infection with Newcastle disease virus. Avian Dis. 8:369-379. Bacon, C. W., and D. Burdick, 1977. Growth of fungi in broiler houses Poultry Sci. 56:65 3—661. Balis, C, and V. Kouyeas, 1968. Volatile inhibitors

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bacteria in the column containing the litter might have been accelerated due to laboratory manipulations. However, even a reduction in the biological activity of these bacteria would still produce enough ammonia in the litter to prevent spore germination in the sensitive species (Table 4). Moreover, adequate inhibitory concentrations of ammonia were found in fresh litter collected for analyses (Table 1). The effect of broiler litter ammonia on spore germination was fungistatic, as spores germinated when they were removed from ammonia and placed in air. A fungitoxic effect was observed during the 12-hr exposure for some species when NH 4 C1 was used to produce concentrations of ammonia exceeding 150/ug/g dry weight of litter. Because these high levels of ammonia were not observed in litter samples, the fungitoxic effect may not occur naturally. The broiler litter used in this study was a composite of the broiler house floor, but there are areas of the floor that are higher in fecal accumulation than others. It is conceivable that there is enough ammonia in these areas to produce the fungitoxic effect. The addition and accumulation of feed and feed additives in litter will also increase the atmospheric ammonia. Chitip is a constituent of one ingredient used in poultry feed and has been shown to increase the levels of ammonia in soils (Schippers and Palm, 1973). The specific broiler management practice, as it relates to house temperature and humidity, would also influence the concentration of ammonia. For example, those houses that use built-up litter and a closed system of ventilation will produce an atmosphere that is high in moisture and atmospheric ammonia. Poultry management conditions leading to high levels of atmospheric ammonia has been criticized, because such conditions have been shown to limit broiler performance directly (Anderson et al, 1964; Valentine, 1964) or indirectly in conjunction with broiler diseases (Quarks and King, 1974; Caveny et al, 1981). To prevent the build-up of harmful levels of ammonia, the addition of substances to litter, e.g., clinoptilolite, and the use of fresh litter are recommended. The level of ammonia indicated as harmful to boilers is between 25 and 50 ppm, but the data in this study indicate that generally, the effective control of spore germination occurs at concentrations that correspond to 10 to 25 jug ammonia/g air. Thus, the level of ammonia within the litter atmo-

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BACON tion of nitrogen from ammonium salts and nitrate by fungi. J. Exp. Bot. 5:232-252. Paulica, D. A., T. S. Hora, J. J. Breadshaw, R. K. Skogerbue, and R. Barker, 1978. Volatiles from soil influencing activities of soil fungi. Phytopathology 68:758-765. Quarles, C. L., and H. F. King, 1974. Evaluation of ammonia and infectious bronchitis vaccination stress on broiler performance and carcass quality. Poultry Sci. 53:1592-1596. Romine, M., and R. Baker, 1972. Properties of a volatile fungistatic factor in soil. Phytopathology 62:602-605. Schefferle, H. E., 1965. The decomposition of uric acid in built-up poultry litter. J. Appl. Bacteriol. 28:412-420. Schippers, B., J. W. Meijer, and J. L. Liem, 1982. The effect of ammonia and other soil volatiles on germination and growth of soil fungi. Trans. Br. Mycol. Soc. 79:253-259. Schippers, B., and L. C. Palm, 1973. Ammonia, a fungistatic volatile in chitin-amended soil. Neth. J. Plant Pathol. 7 9 : 2 7 9 - 2 8 1 . Schippers, B., and F. Pruis, 1978. Fungistatic volatiles released from alkaline soil. Acta Bot. Neerl. 27:156. Thomas, L. C , and G. J. Chamberlin, 1974. The determination of ammonia. Pages 89—97 in Colorimetric Chemical Analytical Methods. 8th ed. The Tinometer Ltd., Salisbury, England. Valentine, H., 1964. A study of the effect of different ventilation rates on the ammonia concentrations in the atmosphere of broiler houses. Br. Poult. Sci. 5:149-159.

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involved in soil mycostasis. Ann. Inst. Phytopathol. Benaki 8 = 145-149. Bremner, J. M., 1965. Inorganic forms of nitrogen Pages 1179-1237 in Methods of Soil Analysis, Part 2, Chemical and Microbiological Properties. C. A. Black, ed. Am. Soc. Agron., Inc., Madison, WI. Caveny, D. D., C. L. Quarles, and G. A. Greathouse, 1981. Atmospheric ammonia and broiler cockerel performance. Poultry Sci. 60:513—516. Dennis, C , and J. M. Gee, 1973. The microbial flora of broiler house litter and dust. J. Gen. Microbiol. 78:101-107. Forgacs, J., H. Koch, W. T. Carll, and R. H. WhiteStevens, 1962. Mycotoxicoses in relationship of toxic fungi to moldy-feed toxicosis in poultry. Avian Dis. 51:363-380. Halbrook, E. R„ A. R. Winter, and T. S. Sutton, 1951. The microflora of poultry house litter and droppings. Poultry Sci. 30:381-388. Keeney, D. R., and D. W. Nelson, 1982. Nitrogeninorganic forms. Pages 643—680 in Methods of Soil Analysis, Part 2. A. L. Page, ed. Am. Soc. Agron., Inc., Madison, WI. Ko, W. H., F. K. Hora, and E. Herlicska, 1974. Isolation and identification of a volatile fungistatic factor from alkaline soil. Phytopathology 64: 1398-1400. Lockwood, J. L., 1977. Fungistasis in soils. Biol. Rev. 52:1-43. Lovett, J., J. W. Messer, and R. B. Jun, 1971. The microflora of southern Ohio poultry litter. Poultry Sci. 50:746-751. Morton, A. G., and A. MacMillan, 1954. The assimila-