A most probable number method for enumeration of rumen fungi with studies on factors affecting their concentration in the rumen

Journal of Microbiological Methods, 16 (1992) 259--270

259

~-) 1992 Elsevier Science Publishers B.V. All rights reserved 0167 - 7012/92/$05.00 MIMET 00534

A most probable number method for enumeration of rumen fungi with studies on factors affecting their concentration in the rumen N.E. Obispo and B.A. Dehority Department of Animal Science, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, USA (Received l May 1992; revision received 22 June 1992; accepted 22 June 1992)

Summary A most probable number (MPN) method was adapted for enumeration of rumen fungi by adding the antibiotics penicillin and streptomycin to the medium to inhibit bacterial growth. The validity of the assay was verified using.a fungicide, cycloheximide, and by comparison to numbers determined in roll tubes. Concentrations of rumen fungi ranged from 1.5 x 10 3 to 1.5 × l 0 6 per g of rumen contents. These concentrations were substantially higher than previously reported ranges. The utilization of both cellulose and soluble carbohydrates as substrates in a single medium allowed simultaneous estimation of total and cellulolytic fungal concentrations. A marked increase in fungal concentrations was observed in steers 1.5 h after feeding, with most of the increase (78%) occurring in the numbers of fungi associated with the solids fraction. Concentrations of tureen fungi in sheep decreased when concentrates were included in the animal's diet. Feeding frequency had little or no effect on the concentration of rumen fungi in steers.

Key words: Anaerobe; Diet effe6ts; Diurnal variation; Most probable number method; Rumen fungi

Introduction A heterogeneous microbial population occurs in the rumen, consisting of bacteria and protozoa along with the more recently discovered anaerobic fungi [1,2]. The bacteria and protozoa have been studied in considerable detail and their contribution to the overall rumen fermentation has been fairly well established; however, our knowledge of the rumen fungi is limited and our understanding of their role in the rumen is not clear. Results from several in vitro studies have shown that the anaerobic rumen fungi are fiber digesters [3-9]. Although these studies have shown that the fungi possess the Correspondence to: B.A. Dehority, Department of Animal Science, Ohio Agricultural Research and Development Center, Wooster, OH 44691-4096, USA.

.:bt required complement of enzymes to degrade cell wall polysaccharides, little is known regarding the magnitude of their activity in the rumen fermentation. Also, there are several inadequacies in the methodology now used to estimate rumen fungal populations. For example, zoospore concentrations have been estimated in filtered rumen fluid by direct microscopic counts or by culture in anaerobic roll tubes; however, zoospores can be associated or entrapped in the rumen particulate matter [10]. In addition, the vegetative stage of the fungal life cycle, the sporangium, is attached to the solids and would not be included in either of these procedures. Thus, the principal objective of this study was to develop a simple and rapid most probable number (MPN) method to estimate population densities of the anaerobic rumen fungi. This in turn would permit studies on factors such as the effect of diet and feeding frequency on fungal concentrations. After the present study was almost completed, Theodorou et al. [11] published an end point dilution procedure for the enumeration of anaerobic fungi. Although their method was based on the MPN technique, it differs in several aspects from the proposed MPN procedure. One major difference is that they used microscopic observation to determine growth. Only those tubes containing motile zoospores, rhizoids and sporangia were scored as positive. Materials and Methods

Animals and diets For those studies concerned with development and evaluation of the MPN method to enumerate anaerobic fungi, rumina! contents were obtained just before feeding from several fistulated steers (average weight 600 kg). The animals were fed once a day (9:00 a.m.) a diet containing 50% chopped alfalfa hay, 50% alfalfa pellets, and a standard supplement of minerals and vitamins. Diurnal variations in total fungal numbers were measured in three fistulated steers (average weight 600 kg), all fed an equal amount daily of the forage diet listed above. Each animal was fed at three feeding frequencies: once; twice; or three times a day. To determine the effects of diet on rumen fungal concentrations, four fistulated sheep (average weight, 65 kg), were used in a 4 x 4 Latin Square study [12]. A basal diet of alfalfa hay was substituted with 20, 50 or 80% whole grain corn. The animals were fed 1.4 kg of each diet once daily (9:00 a.m.) and had free access to water. Composite samples of contents were collected from various locations within the rumen just before the daily feeding. Inoculum preparation The anaerobic cultural techniques were similar to those described by Hungate [13], except for the modifications proposed by Dehority [14]. Procedures for diluting samples were as described by Dehority [14], i.e., 20 g of rumen ingesta were diluted with 180.0 ml of anaerobic dilution solution (ADS) and homogenized for 3 min in a Waring blender under a vigorous strzam O2-free CO2. To evaluate the effect of blending on the viable number of fungi, 20 g of rumen contents were diluted with 180.0 ml ADS in a 250-ml graduated cylinder, and vigorously bubbled with CO2. After gassing for 5 min, this dilution was subsampled and serially diluted in 9.0-ml

261 ADS tubes. The remaining contents in the cylinder were transferred to a blender cup and processed as described above. For estimating the fungal population associated with the fluid and solid portions of rumen contents, 20 ml of rumen fluid, obtained by filtering whole rumen contents through a single layer of polyester cloth (50-/tm pore size, Tetko Inc. Elmsford, NY), were diluted with 180.0 ml of ADS. The mixture was blended and serially diluted as above. Simultaneously, 20 g of whole ruminal contents were processed as described above. Dry matter content was determined for both whole ruminal contents and the fluid fraction. The fluid-associated fungi were estimated by correcting the fluid fraction of rumen contents for the small amount of dry matter contained in the filtered fluid fraction. The fungi associated with the solids fraction were estimated by subtracting the concentration of fungi in the fluid fraction from the concentration of fungi in whole rumen contents. M P N assay

The basal MPN broth used in these studies is listed in Table 1 and is based on the MPN medium described by Dehority et al. [15] for rumen bacteria, l'he final volume of the medium prior to tubing was decreased 14.3% by omitting the appropriate TABLE 1 Composition of the basal MPN broth for rumen fungi Ingredient Mineral solution I (V/V) a Mineral solution II (V/V) a Resazurin solution, 0.1% (v/v) Hemin solution, 0.1% (v/v) Glucose (w/v) Cellobiose (w/v) Maltose (w/v) Xylose (w/v) Trypticase (w/v) Yeast extract (w/v) VFA mixture (v/v) b Rumen fluid (v/v)c Cellulose suspension, 3% (v/v) d Sodium carbonate, 12% (v/v)e Cysteine hydrochloride, 3% (v/v)c Distilled water (v/v) CO2, gas phase

Percentage in medium 15.0 15.0 0.I 0.1 0.I 0.1 0.1 0.1 0.2 0.05 0.45 20.0 25.0 3.33 1.67

5.1 100.0

a Bryant and Burkey [34]. Solution I: 3.0 g K2HPO4/i. Solution II: 3.0 g KH2PO4, 6.0 g (NH4)2SO4,6.0 g NaCI, 0.6 g MgSO4 and 0.6 g CaCI2/I. b Caldwell and Bryant [35]. VFA mixture contains i 7.0 ml acetic acid, 6.0 ml propionic acid, 4.0 ml butyric acid, 1.0 ml isobutyric acid, 1.0 ml n-valeric acid, 1.0 ml isovaleric acid and 1.0 ml ~t-CH3-butyric acid. c Supernatant obtained by straining through a double layer of cheesecloth and centrifuging at 1000 x g for 10 min. d Sigmaceil-20, ball-milled for 24 h (Sigma, St. Louis, MO). e These reagents are tubed anaerobically in small aliquots under nitrogen, sterilized in the tube and stored until used.

amount of distilled water. 6-ml aliquots were tubed (16 x 150 mm tubes) anaerobically and autoclaved. 1 ml of filter-sterilized antibiotic solution was anaerobically added to each tube before use, which brought all the constituents in the medium to the proper final concentration. To evaluate the effect of type of substrate on fungal numbers, either soluble carbohydrates, or cellulose were omitted. The MPN assay procedure was similar to that previously described [15]. For almost all experiments, 10-2-10 -6 dilutions were used to estimate fungal numbers and 10-5-10 - ~1 dilutions for bacterial numbers. Three MPN tubes were inoculated with each dilution. Growth of fungi in MPN tubes containing only soluble carbohydrates was determined after 7 days by increased turbidity or the appearance of flocs and a decrease in pH. Cellulo!ytic fungal concentrations were determined by visible loss of cellulose (generally 75% or more disappearance) and a decrease in pH. When cellulose was included in the medium, a 14-day incubation period was used. The decrease in pH for those tubes with positive fungal growth ranged from 0.2 to 0.5 units.

Antibiotic preparation The antibiotics and concentrations used to inhibit bacterial growth in the medium were those proposed by Akin and Benner [16]; 2000 U of penicillin-G (Sigma, St. Louis, MO) and 130 U of streptomycin sulfate (USB Corporation, Cleveland, OH) per ml of broth. 30/~g Chloramphenicol (Aldrich Milwaukee. WI) per ml of broth, was used to inhibit methanogens and 0.5 mg cycloheximide (USB Cleveland, OH), per ml of broth was used to inhibit fungi [16]. The antibiotics and fungicide, where applicable, were first dissolved in distilled water which had been gassed at least 20 min with oxygen-free CO2 and then sterilized by filtering through a polysulfone membrane (Gelman Sciences, Ann Arbor, MI) with a pore size of 0.2/~m. I ml of this filtersterilized antibiotic solution was added aseptically and anaerobically to each tube of M PNI . . . . .

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Verification procedure Roll tube medium used to validate the MPN procedure was identical to that listed in Table 1, with all substrates except glucose (0.1%) omitted and 1.83% agar added. Procedures were similar to those described by Grubb and Dehority [17] and Joblin [i 8]. Two additional antibiotics were used to control bacterial growth in some of the assays; oxytetracycline at 0.25 mg/ml of medium and neomycin sulfate at 0.3 mg/ml of medium (Sigma) [19]. Initially, roll tubes were counted using an inverted microscope at 125 x magnification. However, with experience, it was subsequently possible to distinguish between colonies of bacteria and fungi using a dissecting microscope.

Statistics The data were transformed to log~0 because the logarithmic distribution tends tq be more symmetrical than the distribution of the estimated density values [20]. Statistical treatment of the data was performed by analysis of variance, with mean

263 separation by the method of least significant difference, and paired t-test [12]o Results

Effects of adding antibiotics and fungicide, singly or combined, to the basal MPN medium for enumeration of both total and cellulolytic rumen microorganisms is shown in Table 2. Although there was a slight decrease in apparent bacterial numbers when the fungicide was added to the basal medium, the decrease was not significant. Apparent fungal numbers were lower (p<0.05) than the total microbial numbers. Microscopic observations of the contents of the MPN tubes containing antibiotics confirmed the presence of both zoospores and sporangia. Lack of growth in the MPN tubes when the medium was treated with both antibiotics and fungicide would substantiate the selective growth of these two different populations. Higher concentrations of total fungi (p < 0.05) were observed in media containing soluble carbohydrates with or without cellulose, as compared to cellulose alone. Cellulolytic fungal numbers also increased (p<0.05) when soluble carbohydrates were included in the medium with cellulose. Using both substrates, soluble carbohydrates and cellulose, it should be possible to simultaneously estimate both total and cellulolytic fungal numbers in a single medium, similar to the procedure of Dehority et al. [15] for anaerobic bacteria. Blending the rumen contents for 3 min did not affect the number of viable anaerobic fungi. The mean log~0 concentrations + S.E. were 4.9 + 0.41 without blending and 5.0 + G.37 with blending. It was observed that after blending, the coarse plant materials in the sample were reduced to a more uniform size. To verify the accuracy of the proposed MPN procedure, fungal numbers were simultaneously estimated in roll tubes. Considerable bacterial growth occurred in roll tubes contain~_ag penicillin and streptomycin at the same concentrations used in the MPN medium, and fungi had to be counted with a microscope. Less than 10% of the total colonies present in the roll tubes were fungi (Table 3); however, numbers were TABLE 2 Use of antibiotics and a fungicide in an MPN assay for rumen fungi Additions to basal m e d i u m

Log~0 microorganisms per g rumen contents TotaP

None Antibiotics (fungal numbers) a ~ Fungicide (bacterial numbers) ° Antibiotics + fungicide

Cellulolytic

Mean

S.E.

Mean

S.E.

9.38 d 4.39 e 9.13 d 0.00 f

0.00 0.14 0.38 0.00

8.19 d 3.66 e 7.42 d 0.00 f

0.40 0.3 l 1.24 0.00

Penicillin and streptomycin added. b Cycloheximide added. c For all treatments except antibiotics + fungicide, total numbers were higher (p <0.01) than cellulolytic numbers. a,e.f Means in the same column with different superscript letters differ (p < 0.05). a

264 TABLE 3 Comparison of rumen fun~ concentrations determined with roll tubes and the MPN procedure MPN assay

Roll tube assay

Log~0 fungi per g rumen contents

Log10 fungi per g rumen contents

Percentage of total colonies

4.63 3.63 4.63 4.38 4.38 4.33 S.E. 0.18

4.1 i 3.52 4.63 4.10 4.84 4.24 0.23

10.3 6.6 6.3 2.0 10.8 7.2 1.59

not different from those estimated with the MPN assay (p> 0.5). Several additional antibiotics, previously used by Lowe et al. [19], were added to the roll tube medium in an attempt to inhibit bacterial growth. Both chloramphenicol and oxytetracycline appeared to completely inhibit bacterial growth in roll tubes; however, fungal numbers were also decreased (Table 4). Neomycin was ineffective in lowering bacterial numbers, but did decrease fungal numbers. The individual antibiotics, in conjunction with penicillin and streptomycin, decreased mean fungal numbers as follows: chloramphenicol, 28%; oxytetracycline, 82%; and neomycin, 64%. Since the addition of chloramphenicol completely inhibited bacterial growth and only decreased fungal numbers by 28%, it was evaluated in both the M P N and roll tube assays. Results of this study are shown in Table 5, and adding chloramphenicol significantly reduced fungal numbers in both procedures: 70% in the MPN assay and 37% in the roll tube assay. As shown in Table 3, the MPN and roll tube assays were not different using only penicillin and streptomycin. v n me oasis of me above data, it was concluded that the MPN assay, using f'~__

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TABLE 4 Differences in fungal concentrations in roll tubes resulting from the addition of chloramphenicol, oxytetracycline or neomycin to a basal medium containing penicillin and streptomycin Fungi per g o f rumen contents "~ P and S b

P, S and Ch

P, S and Ox

P, S and N

25000 (5.0) c TMC d 99000 (3.2) 04 200 ( T M C ) = 56 100

21 000 (100) 59000 (100) 51 400 (100) 30000 (100) 40 400

0 16000 (100) 12000 (100) 13000 (100) 10 250

10700 7 200 32 700 30000 20 150

(TMC) (TMC) (3.3) (TMC)

~ Determined in roll tubes. b Antibiotics added to basal medium: P = penicillin; S = streptomycin; Ch = chloramphenicol; Ox = oxytetracycline; N = neomycin. ¢ Percentage of total colonies in roll tubes. d T M C = too many to count.

265 TABLE 5 Comparison of fungal concentrations determined in roll tubes and by the MPN assay using medium with and without chloramphenicol Loglo fungi per g rumen contents MPN assay

Roll tube assay

P and S"~

P, S and Ch b

P and S

P, S and Ch

-x .A. .~c . SE 0.15

4.18 d 0.09

4.44 ~ 0.13

4.20 f 0.15

" Penicillin and streptomycin. b Penicillin, streptomycin and chloramphenicol. c,d Means under the same heading differ significantly at p < 0.06. c e Means are not different (p >0.1). e'f Means under the same heading differ significantly at p < 0.05. penicillin a n d s t r e p t o m y c i n to inhibit bacterial g r o w t h , gave results c o m p a r a b l e to t h o s e o b t a i n e d in roll tubes. Results r e p o r t e d in the r e m a i n d e r of this study were o b t a i n e d using the M P N m e t h o d . T h e n u m b e r o f a n a e r o b i c fungi a s s o c i a t e d with the fluid a n d p a r t i c u l a t e fractions in steer c u m e n c o n t e n t s was e s t i m a t e d at 0, 1.5 a n d 3.0 h after feeding (Fig. 1). T h e solids associated fungi ( S A F ) c o n s t i t u t e d 86.0, 77.2, a n d 6 9 % of the total fungi ( T F ) at 0, 1.5 a n d 3.0 h after feeding, respectively, while the fluid associated fungi ( F A F ) c o n s t i t u t e d 14.0, 22.8 a n d 31.0%. C o m p a r e d to 0 h, there was a n increase of 145% for

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266 TABLE 6 Effect of diet on the concentration of rumen fungi Log~0 fungi per g rumen contents

Concentrate" (%)

0 20 50 80

Mean

S.E.

5.55 b 4.82 c 5.33 d 5.30 d

0.28 0.32 0.20 0.30

" Percentage of whole grain corn included in the basal alfalfa hay diet. ~,.c.d Means in the same column with different superscript letters differ (t9 <0.01).

the SAF and 346% for the F A F at 1.5 h post-feeding, which represented an overall increase of 173 % for the TF. A decrease in the TF of 51% occurred between 1.5 and 3.0 h, which primarily occurred in the SAF fraction. Substitution of corn for hay in the diet of sheep resulted in a decrease in fungal numbers (p<0.01) as compared to the all hay diet (Table 6). Surprisingly, fungal numbers in animals fed the 20% corn level were lower (p<0.01) than when 50 and 80% corn were fed. In general, rumen fungal concentrations remained fairly constant over a period of 24 h, and did not appear to be markedly influenced by feeding frequency. Data for one of the steers are shown in Fig. 2, and some variation can be observed when the 4

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Fig. 2. Effects of feeding frequency on diurnal variation of rumen fungi numbers, animal no. 1. Vertical arrows denote feeding times.

267 animal was fed twice or three times a day. Similar variation was noted with the once a day feeding in animal 2 and three feedings per day in animal 3, however, no consistent pattern was observed. Statistical analysis of the data (result~ not shown) indicated that there were no significant.differences between animals, sampling time or feeding frequency. Discussion

For this study the fungal population has been designated as the number of fungi or fungal numbers per g of rumen contents. Presumably, this includes sporangia plus free and attached zoospores. Several investigators have demonstrated that the suppression of bacterial growth by the addition of the antibiotics (penicillin and streptomycin) to the media provides ar~ appropriate method to enumerate fungi [16,21-23]. The present results would substantiate this conclusion. The levels of penicillin and streptomycin used in the roll tube medium apparently are not bactericidal for all rumen bacteria. Small, slow growing bacterial colonies, ranging in concentration from 5-70 x 10 4 per g of rumen contents, were present in the roll tubes. This is markedly lower than the normal bacterial concentrations found in rumen contents, i.e., about 1 x 101° per g. It would appear that the growth of these bacteria is sufficiently inhibited in MPN broth so that they do not interfere in the results. This is seen quite readily in Table 2, where microorganism numbers are significantly decreased in the presence of the antibiotics, not affected by the fungicide and no growth at all occurs when the antibiotics and fungicide are combined. Although roll tube procedures could be used to enumerate rumen fungi, the procedure is time-consuming and requires significant training and skill for both inoculation and subsequent reading of the tubes under the microscope. The ease and simplicity of the MPN procedure would make it the method of choice for routine USe.

The observed range in fungal concentrations in the present study was 1.5 × 10 3 1.5 x 10 6 per g of rumen contents. Reported concentrations from previous studies, estimated either by direct count or with anaerobic roll tubes, were generally in the lower half of this range [18,24,25]. However, it should be noted that most other studies used rumen fluid as their inoculum, which means that any fungi attached to particulate matter would not have been counted (Fig. 1). In evaluating substrates for the MPN analysis, concentrations estimated with only cellulose as substrate were significantly lower than those with soluble carbohydrates or the combination of cellulose and soluble carbohydrates. The logical conclusion might be that only a portion of the total number were cellulolytic; however, the number of cellulolytic fungi increased and was not different from the total when both soluble carbohydrates and cellulose were included as substrate~. Although most strains of fungi studied in detail are cellulolytic, a few have been isolated which are unable to grow in media containing only purified cellulose as an energy source [26,27]. Previous studies by Orpin and coworkers have indicated that the viability of the zoospores appears to depend on availability of some readily fermentable soluble carbohydrate in the medium [28,29]. This might explain the present results, i.e., more

268 zoospores germinate in the soluble carbohydrate containing medium. When enumerating rumen fungi it is important to take into account that the population consists of two or possibly three biological entities which are located in distinct niches in the rumen contents; the free zoospore (present in rumen fluid), zoospores attached to particulate matter and sporangia, which are also attached to particulate matter. By means of a filtration procedure, based on the differences in size between the free zoospores, attached zoospores and sporangia, free and attached fungi were estimated. Total concentrations of fungi, both free and attached to the solids, increased by 173% at 1.5 h as compared to the same population at 0 h. This increment at 1.5 h represented an actual increase of 145 and 346% over 0 h concentrations for the SAF and FAF, respectively. These data would clearly confirm the release of zoospcres from the sporangia at feeding, which is in agreement with the previous observations of Orpin [30]. Assuming attached zoospores would be included in the total count, no obvious explanation is available for the marked decrease in fungal concentrations between 1.5 and 3.0 h. Since the animals continued to eat and drink from 0 to 3 h, dilution may be the explanation for the decrease; however, the increase between 0 and 1.5 h actually would then probably be larger than measured. The concentration of fungi per g of rumen contents decreased when the proportion of concentrate in a basal diet of forage was increased from 0 to 20%. This observation is consistent with results found by other authors who estimated the fungal numbers from animals fed different types of diets [31-33]. However, the subsequent increase in the concentration of fungi as the proportion of concentrate in the diet increased from 20 to 50 and 80% was unexpected. Further investigations appear to be required to understand the cause(s) of these differences. In general, fungal numbers tended to remain fairly constant over a period of 24 h regardless of the feeding frequency. Although some variations were observed between individual animals and feeding frequencies during these studies, there were no consistent patterns which could be attributed to the number of times the animal ~.~e

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Acknowledgemeats Salaries and support for this work were provided by state and federal funds appropriated to The Ohio Agricultural Research and Development Center, The Ohio State University. The senior author (N.E.O.) was supported by FONAIAP, Venezuela. We thank Pat Tirabasso for technical assistance in comparison of the MPN and roll tube procedures.

Referenccs 1 Hungate, R.E. (1950) The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14, 1-49. 2 0 r p i n , C.G. (1975) Studies on the rumen flagellate Neocallimastixfrontalis. J. Gen. Microbiol. 91, 249-262. 3 Bauchop, T. and Mountfort, D.O. (1981) Cellulose fermentation by a rumen anaerobic fungus in both the absence and presence of rumen methanogens. Appl. Environ. Microbiol. 42, 1103-1110. 4 Gordon, G.L.R. and Ashes, J.R. (1984) In vitro digestion of wheat straw by different rumen anaerobic fungi. Can. J. Anim. Sci. 64 (Suppl.), 156-157.

269 5 H6braud, M. and F~vre, M. (1988) Characterization of hydrolases produced by rumen anaerobic fungi. Reprod. Nutr. Develop. 28, 131-132. 6 Lowe, S.E., Theodorou, M.K. and Trinci, A.P.J. (1987) Cellulases and xylanase of an anaerobic rumen fungus grown oq wheat straw, wheat straw holocellulose, cellulose, and xylan. Appl. Environ. Microbiol. 53, 1216-1223. 7 0 r p i n , C.G. and Letcher, A.J. (1979) Utilization of cellulose, starch, xylan, and other hemicelluloses for growth by the rumen phycomycete Neocallimastix frontalis. Current Microbioi. 3, 121-124. 8 0 r p i n , C.G. and Hart, Y. (1980) Digestion of plant particles by rumen phycomycete fungi. J. Appl. Bacteriol. 49, x. 9 Pearce, P.D. and Bauchop, T. (1985) Glycosidases of the rumen anaerobic fungus Neocallimastix fronta!is grown on ,-,~11,,I,,~i,,substrates, t~,,nl lz,,,,~,.,-,,, Microbio!. ao l-~t,~_~'~,~o 10 Orpin, C.G. and Joblin. K.N. (1988) The rumen anaerobic fungi. In: The Rumen Microbial Ecosystem (Hobson, P.N., Ed.), pp. 129--150, Elsevier, London, New York. !1 Theodorou, M.K., Gill, M., King-Spooner, C. and Beever, D.E. (1990) Enumeration of anaerobic Chytridiomycetes as thallus-forming units: Novel method for quantification of fibrolytic fungal populations from digestive tract ecosystem. Appl. Environ. Microbioi. 56, 1073-1078. i 2 Steel, R.G.D. and Torrie, J.H. (1960) Principles -and procedures of statistics. McGraw-Hill Book Co., NY. 13 Hungate, R.E. (1966) The rumen and its microbes. Academic Press, New York. 14 Dehority, B.A. (1969) Pectin-fermenting bacteria isolated from the bovine rumen. J. Bacteriol. 99, 189-196. 15 Dehority, B.A., Tirabasso, P.A. and Grifo, A.P. Jr. (1989) Most-probable number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium. Appl. Environ. Microbiol. 55, 2789-2792 16 Akin, D.E. and Benner, R. (1988) Degradation of polysaccharides and lignin by ruminal bacteria and fungi. Appi. Environ. Microbiol. 54, 1117-1125. 17 Grubb, J.A. and Dehority, B.A. (1976) Variation in colony counts of total viable anaerobic rumen bacteria as influenced by media and cultural methods. Appl. Environ. Microbiol. 31,262-267. 18 Joblin, K.N. (1981) Isolation, enumeration, and maintenance of rumen anaerobic fungi in roll tubes. Appl. Environ. Microbiol. 42, 1119-1122. 19 Lowe, S.E., Theodorou, M.K., Trinci, A.P.J. and Hespell, R.B. (1985) Growth of anaerobic rumen fungi on defined and semi-defined media lacking rumen fluid. J. Gen. Microbiol. 131, 2225-2229. 20 Cochran, W.G. (1950) Estimation of bacterial densities by means of the most probable number. DIUIIIULII~.~3

,

IU..)--I

IO.

2 i Akin, D.E. and Rigsby, L.L. (1987) Mixed fungal populations and iignocellulosic tissue degradation in the bovine rumen. Appl. Environ. Microbiol. 53, 1987-1995. 22 Joblin, K.N., Campbell, G.C., Richardson, A.J. and Stewart, C.S. (1989) Fermentation of barley straw by anaerobic rumen bacteria and fungi in axenic culture in a co-culture with methanogens. Left. Appl. Microbiol. 9, 195-197. 23 Windham, W.R. and Akin, D.E. (1984) Rumen fungi and forage fiber degradation. Appl. Environ. Microbiol. 48, 473-476. 24 Orpin, C.G. (1974) The rumen flagellate Callimastix frontalis: Does sequestration occur? J. Gen. Microbiol. 84, 395-398. 25 Ushida, K., Tanaka, H. and Kojima, Y. (1989) A simple in situ method for estimating fungal population size in the rumen. Lett. Appl. Microbiol. 9, 109-111. 26 Phillips, M.W. and Gordon, G.L.R. (1988) Sugar and polysaccharide fermentation by rumen anaerobic fungi from Australia, Britain and New Zealand. BioSystems 21,377-383. 27 Gordon, G.L.R. and Phillips, M.W. (1989) Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen. Appl. Environ. Microbiol. 55, 1703-1710. 28 Orpin, C.G. and Bountiff, L. (1978) Zoospore chemotaxis in the rumen phycomycete Neocallimastix frontalis. J. Gen. Microbiol. 104, 113-122. 29 Orpin, C.G. and Greenwood, Y. (1986) Nutritional and germination requirements of the rumen Chytridiomycete Neocallimastix frontalis. Trans. Br. Mycol. Soc. 86, 103-109. 30 Orpin, C.G. (1977) On the induction of zoosporogenesis in the rumen phycomycetes Neocallimastix

270 frontali,~. Piromonas communis, and Sphaeromonas communis. J. Gen. Microbiol. I 01, ! 8 I- 189. 3! Akin, D.E., Borneman, W.S. and Windham, W.P. (1988) Rumen fungi: Morphological types from Georgia cattle and the attack on forage cell walls. BioSystems 21,385-391. 32 Grenet, E., Breton, A., Fonty, G., Barry, P. and R~mond, B. (1988) Influence of the diet on rumen anaerobic fun~. Reprod. Nutr. D6veiop. 28, 127-128. 33 Grenet, E., Fonty, G., Jamot, J. and Bonnemoy, F. (1989) Influence of diet and monesin on development of anaerobic fungi in the rumen, duodenum, cecum, and feces of cows. Appl. Environ. Microbiol. 55, 2360-2364. 34 Bryant, M.P. and Burkey, L.A. (1953) Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J. Dairy Sci. 36, 205-217. 35 Ca!dwe!i, D.R. and Bryant, M.P. (1966) Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Appl. Microbiol. 14, 794-801.