Vol. 38, No. 1APPUED AND ENVIRONMENTAL MICROBIOLOGY, JUly 1979, p. 148-1580099-2240/79/07-0148/11$02.00/0

Rumen Anaerobic Fungi of Cattle and SheepT. BAUCHOP

Applied Biochemistry Division, Department ofScientific and Industrial Research, Palmerston North,New Zealand

Received for publication 9 April 1979

Plant fragments obtained from natural rumen digesta of fistulated cattle andsheep were examined by scanning electron microscopy. Various plant materialssuspended in the rumen for different times were examined likewise. By 2 h largenumbers of phycomycetous fungal zoospores were found attached to fibrous plantfragments, particularly vascular tissues. The subsequent development of thesefungi resulted in production of thalli with extensive rhizoids and with sporangiaup to 175 tum long. Scanning electron microscope examination of plant fragmentsrandomly selected from natural rumen contents of both cattle and sheep dem-onstrated widespread colonization by large populations of these anaerobic fungi.Furthermore, all plant fragments suspended in nylon bags in the rumen were alsoextensively colonized. These findings demonstrate that plant fragments in therumen are the sites of colonization and development by the anaerobic phycomy-cetous fungi. In addition, the results suggest that these fungi may form asignificant part of the rumen microbiota in cattle and sheep fed on fibrous dietsand suggest that they may be important in fiber digestion.

Orpin was the first to demonstrate (4, 5, 7)that some of the sheep rumen microorganismsbelieved to be protozoan flagellates were in factzoospores of anaerobic phycomycetous fungi.These fungi, however, remain classified as pro-tozoa (4, 5, 7).

Initially Orpin (4) isolated a multiflagellatedorganism, which he named Neocallimastix fron-talis and which possessed a life cycle alternatingbetween a motile flagellated form and a "non-motile vegetative, reproductive form." Posses-sion of a rhizoid by the reproductive body led tothe suggestion (4) that the organism might be afungus rather than a protozoan. Additional "flag-ellates", named Sphaeromonas communis (5)and Piromonas communis (7), were isolatedlater, and a resemblance to aquatic phycomy-cetous fungi was noted (5). After this, N. fron-talis was described as a rumen phycomycete forthe first time (6), and invasion of plant materialssuspended in nylon bags in the sheep rumen wasdescribed. Orpin found that when zoospores didattach, they showed a preference for inflores-cence tissue from feed materials (6, 7, 9). How-ever, N. frontalis was found only rarely attachedto food particles in filtered rumen contents (6),and it was not possible to determine whethersporangia present in whole rumen contents wereattached to particles (6). Our understanding ofthe relationships of these fungi to rumen digestaplant fragments has remained uncertain, as in-dicated by the recent statement (9) that "zoo-

spores may germinate upon and grow at theexpense of plant tissue present in the rumen."Because zoospores of the rumen phycomy-

cetes had been found to be present in low num-bers in strained rumen fluid (4, 5, 7), they werebelieved to be unimportant members of the ru-men microbiota. Sporangia of these fungi hadbeen found mainly free in strained rumen fluid,and it was believed that they could exist in thatstate in rumen contents (4, 5, 7, 8). However, inview of the low count of sporangia (up to 3.6 x104/ml) in strained rumen fluid, it was concludedthat the overall effect of these fungi on rumenmetabolism was probably not great (8).

During a scanning electron microscope (SEM)investigation of microbial digestion of plant ma-terials suspended in the sheep rumen, large num-bers of spherical to ovoid bodies (6 to 10 ,tm)were found to attach rapidly to exposed vascularcylinders of partially degraded lucerne stems.Their identity was revealed by the occasionalfinding on the plant pieces of similar but flagel-lated forms that corresponded to the descrip-tions of some of the zoospores of the rumenanaerobic phycomycetous fungi given by Orpin(4, 5, 7). The attached nonflagellated forms werethen identified as an early developmental stagein the life cycle of these fungi. These findingssuggested a central role for fibrous plant mate-rials in the life cycle of these fungi in the rumenand prompted a search for them on naturalrumen digesta of cattle and sheep.

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In the present study, fibrous plant fragmentswere shown to be the principal sites of coloni-zation by the rumen anaerobic fungi. The rapidattachment of large numbers of zoospores, to-gether with the demonstration of extensive pop-ulations of thalli attached to plant fragments,suggests that the rumen phycomycetes mayform an important part of the rumen microbiota.

MATERIALS AND METHODSAnimals. Romney Marsh wether sheep (Ovis ar-

ies), each fitted with a rumen cannula, were kept inmetabolism crates and fed once daily. Three sheepreceived pelleted lucerne (Medicago sativa L.), and asecond group of three received chaffed lucerne. Fourcattle (Bos taurus), steers and cows, each fitted witha rumen cannula, were maintained outdoors on asawdust loafing pad with free access to water and salt.During experiments they were taken indoors for feed-ing and received meadow hay ad libidum between 0900and 1500 h.

Natural rumen digests. Samples of whole rumencontents, usually obtained after overnight fasting,were fixed with an equal volume of 4% (wt/vol) un-buffered formaldehyde. Stem or leaf samples wereremoved by using forceps, washed thoroughly by agi-tating in normal saline, and then transferred to 4%(wt/vol) unbuffered formaldehyde to await furtherprocessing for SEM or light microscopy.

Plant fragments. Stem vascular cylinder tissue offresh or chaffed lucerne was prepared by gently shav-ing off the outer tissues of the stem with a razor blade.Sisal (vascular bundles surrounded by schierenchy-matous sheaths from leaves ofAgave sisalona L.) wascommercially available as twine. Pieces of other plantmaterials approximately 20 mm long were obtainedfrom chaffed lucerne, meadow hay, and wheat strawand from fresh plants.

Pieces of plant material suspended in a nylon bagin the rumen were removed at timed intervals, rinsedin normal saline, and fixed in 4% (wt/vol) unbufferedformaldehyde.SEM. After fixation, stem samples from the rumen

were rinsed three times with distilled water, blotteddry, and freeze dried (1). Other details of samplepreparation for SEM examination were as describedpreviously (1).

Light microscopy. Pieces of stem or stem vascularcylinder from the rumen were sliced longitudinallywith a razor blade. Thin slices were stained with 0.1%(wt/vol) trypan blue in lactophenol. Leafsamples werestained without slicing. Stained samples were exam-ined by bright-field illumination.

Culture of fungi. Fungi were grown anaerobicallyat 39°C in tubes of medium prepared by the methodof Hungate (3). The gas phase was CO2.

Salt solutions (2) were used in medium preparation.Solution A contained (percent, wt/vol): KH2PO4, 0.3;NaCl, 0.6; (NH4)2SO4, 0.3; CaCl2, 0.03; and MgSO4,0.03. Solution B contained 0.3% (wt/vol) K2HPO4.The medium used was modified from Orpin (4) and

had the following composition: solution A, 16.5 ml;solution B, 16.5 ml; cell-free ovine rumen fluid, 17 ml;

distilled water, 50 ml; NaHCO3, 0.5 g; yeast extract(Difco), 0.1 g; peptone (Difco), 0.1 g; glucose, 0.3 g;agar, 0.1 g; cysteine-HCl, 0.02 g; and resazurin, 0.0001g. Medium (7 ml) was tubed in Hungate tubes (BellcoGlass, Inc.) and sterilized by autoclaving. On coolingto room temperature, 0.35 ml of antibiotic solution(benzylpenicillin, 2 x 104 IU/ml and streptomycinsulfate, 2 mg/ml) was added.

Inoculum was 0.35 ml of fresh rumen contents,strained through cheesecloth.

RESULTSOvine rumen fungi. In each study where

animals consumed appreciable amounts of fiber(pelleted or chaffed lucerne), large numbers offungal sporangia were found attached to stemfragments selected at random from rumen con-tents of sheep before morning feeding (Fig. lathrough c). Highest numbers were found withthe chaffed lucerne diet, which contained morestalky material than the pelleted lucerne diet.The presence of fungi at different stages of

development on the same piece of plant material(Fig. la and b) demonstrated that fungal infec-tion could occur at widely differing times of theday. Fig. lc shows a heavy population of fungiat an early stage of development. In each ofthese samples (Fig. la through c) the fungi wereattached to the vascular cylinder of the lucernestem; the outer, thin-walled tissues of the stemwere already removed by this stage of digestion.Several examples were also obtained where por-tions of fungal rhizoids ("mycelium") remainedattached, even when exposed (Fig. 2a and b)after digestion of stem thin-walled tissues.

Lucerne leaves in the chaffed diet were exten-sively digested after 24 h in the rumen, butfragments also had fungi attached (Fig. 3),mainly to the stalks and ribs.

Zoospores were always present in rumen con-tents of sheep receiving pelleted or chaffed lu-cerne diets, and inoculation of fresh rumen fluidinto anaerobic medium produced rich culturesof characteristic (4) thalli within 48 h.Phycomycetous fungal sporangia or zoospores

were not detected microscopically in rumen con-tents of sheep on diets low in fiber, e.g. contin-uously grazed pasture that did not have theopportunity to develop stalks or seedheads. At-tempts to culture anaerobic fungi from rumencontents of such sheep were also unsuccessful.These rumen contents contained only soft leafymaterial, and after starvation of sheep for 24 h,contents were very liquid and contained littlefibrous material.Bovine rumen fungi. The meadow hay diet

used in this study contained large amounts ofstems, mainly of mixed grasses but with smallquantities of red clover (Trifolium pratense L.).

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Cs~FIG. 1. Sporangia of rumen phycomycetes attached to vascular cylinder of lucerne stem from natural

rumen digesta of sheep. Samples were obtained before feeding in the morning. (a) and (b) Sporangia at early(arrows) and late stages ofdevelopment. (c) A large population at an early stage in development ofsporangia.

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FIG. 2. Thalli of rumen phycomycetes with part of rhizoids remaining attached to lucerne stem vascularcylinder after soft stem tissues have been digested. (b) also shows many sporangia (arrows) at different stagesin development from zoospores. Samples were from natural rumen digesta of a sheep and were obtainedbefore feeding in the morning.

FIG. 3. Sporangia of rumen phycomycetes attached to the main rib of a leaf from lucerne chaff in thenatural rumen digesta of a sheep. The sample was obtained before feeding in the morning. Mesophyll tissuewas extensively digested by this time.

Red clover stem digesta fragments, removedfrom rumen contents before feeding in the morn-ing, had large numbers of fungi attached to them(Fig. 4). As with lucerne stem in sheep, thesofter, outer tissues were digested away by thisstage, and the fungi remained attached to themore resistant vascular cylinders.

Fungal colonization on grass stems in meadowhay possessed several features not observed withother feed materials. The highest fungal popu-lations were always found on the inner surfaceof the hollow stem (Fig. 5). A range of sizes ofsporangia was present, indicating different timesof fungal colonization, but on the basis of spor-

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FIG. 4. Sporangia of rumen phycomycetes attached to the vascular cylinder of a red clover stem from thenatural rumen digesta of a steer before feeding in the morning.

FIG. 5. Sporangia of rumen phycomycetes attached to the inside surface of the hollow stem of a grass fromthe natural rumen digesta of a cow. The sample was obtained before feeding in the morning.

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angial morphology few types appeared to bepresent in this niche. In contrast, although fewfungi colonized the outer surface of grass stems,there was a greater variety of sporangia present.The long cylindrical form (Fig. 6a and b) maybe identical to that present on red clover stem(Fig. 4). Sharply pointed sporangia (Fig. 6c) andlong thin sporangia (Fig. 6d) have been foundonly on the outer surfaces of grass stems inbovine rumens.Fungal colonization of plant materials

suspended in the rumen. Pieces of lucernestem in nylon bags were suspended in the ru-mens of sheep, and samples were removed atintervals for examination by SEM. At 2 h largenumbers of spherical to ovoid bodies (6 to 10

Mm) (Fig. 7a and b) were found attached toexposed vascular cylinders. A few similar butflagellated forms were also found on stem vas-cular cylinders (Fig. 8). On the basis of mor-phology, these were identified as zoospores ofrumen anaerobic phycomycetous fungi. The at-tached unflagellated forms were then identifiedas an early developmental stage in the life cycleof these fungi. Apparently flagellae separatedsoon after attachment of the zoospores to planttissues. Extensive zoospore attachment wasrapid, and colonization in high numbers wasindicated by the count of 10" sporangial earlydevelopmental forms per cm' (counted on anarea of 0.14 mm2) (Fig. 9) on the surface of thestem vascular cylinder.

E.AI

FIG. 6. Different sporangial forms ofrumenphycomycetes on the epidermis ofa grass stem from the naturalrumen digesta of a cow. Samples were obtained before feeding in the morning.

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- -_ 9FIG. 7. Early stage in the development of sporangia from zoospores of rumen phycomycetes. Attached to

the vascular cylinder of a lucerne stem suspended in a sheep rumen for 2 h.FIG. 8. Zoospore of rumen phycomycete on the vascular cylinder of a lucerne stem suspended in a sheep

rumen for 2 h.FIG. 9. Colonization by rumen phycomycetes on the vascular cylinder of a lucerne stem suspended in a

sheep rumen for 2 h. Early stage of sporangial development from zoospores.

The presence of fungal sporangia or rhizoids different time intervals. Plant materials studiedafter rapid colonization by zoospores was ob- in sheep included sisal fiber, stems of fresh lu-served with several different plant materials sus- cerne, chaffed lucerne and wheat straw, vascularpended in the rumens of cattle and sheep for cylinders from stems of fresh and chaffed lu-

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cerne, and leaves from chaffed lucerne andwheat straw. In cattle, plant materials investi-gated included stems of red clover and grass inmeadow grass hay and leaves of wheat strawand grass in meadow grass hay. The vascularcylinders of stems and the vascular bundles andadjacent mesophyll tissue in leaves were theprincipal regions of colonization. Grass stemswere colonized mainly on the inside surface ofthe hollow stem.Rumen incubation periods of 24 to 48 h were

required for development of large sporangia onsisal fiber (Fig. 10a and b). With wheat strawstems, fungal colonization was low, and largesporangia developed even more slowly (2 to 4days). Every piece of all of the other plant ma-terials suspended in the rumen at different timesof day was extensively colonized by fungi, andby 24 h many large sporangia were always foundattached to the surfaces (Fig. lla through c).These experiments were repeated many times inboth cattle and sheep with several pieces ofindividual plant material on each occasion. Withevery piece of these plant fragments suspendedin the rumens extensive fungal colonization wasobtained.Fungi in feed. The possibility that some of

the fungi were derived from feed was examined.With plant fragments stained with trypan blue,fungal hyphae were frequently detected in thedried feeds studied, but sporangia similar tothose found on plant fragments from the rumnenwere never observed. Attempts to culture rumenfungi from feed material proved negative also,although the anaerobic phycomycetes werereadily isolated from rumen contents of bothcattle and sheep. Furthermore, sterile (auto-

claved) wheat straw leaves suspended in therumen were rapidly colonized by the anaerobicphycomycetous fungi.

DISCUSSIONThe rumen anaerobic phycomycetous fungi

have now been shown to extensively colonizeplant materials in rumen natural digesta. Thepresent demonstration of rapid colonization andattachment by large numbers of zoospores torumen fibrous plant fragments, with subsequentdevelopment of thalli, resolves much of the con-fusion surrounding the understanding of the lifecycle of these fungi in the rumen. In majormeasure, this confusion has been the result ofthe practice, common in studies ofrumen micro-biology, of using strained rumen contents (4-9)and discarding the rumen solids. This paper hasshown that the commonly discarded solids frac-tion contains large numbers of the vegetativeand reproductive stages of the rumen phyco-mycetes.

Zoospores of phycomycetous fungi character-istically attach to fragments of biological mate-rials, where development of rhizoids can occurin or on the substratum. The evidence presentedin this paper demonstrates that this is the casealso with the rumen phycomycetes. Stems ofseveral species of pasture plants and leaves ofgraminaceous plants are the main substrates forfungi in the rumen. Ultimately the fungi werefound closely associated with the more slowlydigested fractions, the vascular tissues of stemsand leaves.Although the fungi found on stems were

mainly attached to vascular cylinder tissue, thisdoes not mean that hyphae do not invade softer

FIG. 10. Sporangia of rumen phycomycetes attached to sisal twine (vascular tissue) suspended in a sheeprumen for 48 h. (a) Cut end. (b) Side of piece of twine with attached sporangia at early (arrows) and latestages of development.

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FIG. 11. Sporangia ofrumen phycomycetes attached to stem offood fragments suspended in a rumen for 24h. (a) Cut end of lucerne stem from a sheep. (b) Cut end of red clover stem from a cow. (c) Inside surface ofhollow stem ofgrass from a cow.

tissues also. Exposure of fungal rhizoids on theoutside of the lucerne vascular cylinder (Fig. 2aand b) indicates that thin-walled tissue, in thiscase cortex, is also invaded. This is indicatedalso in several examples (Fig. la and 4) whereconsiderable lengths of hyphae were found out-side the vascular cylinder, with sporangia thusappearing on stalks. The length of this exposedstalk may represent the thickness of thin-walledtissue through which the fungus has pen-

etrated before reaching the vascular cylinderafter initial zoospore colonization at the epider-mal surface of the stem. In contrast, sporangiawhich are directly attached to the vascular cyl-inder probably represent colonization by zoo-spores at a later stage in the digestion of theplant fragments.The major route of fungal invasion of all plant

fragments studied was via damaged tissue. Themain exception was in the case of grass stems,

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where the inner surface of the hollow stem wasthe major site of colonization. But as with dam-aged tissue sites, this surface also is an exposedinterior tissue. A few fungi were capable of in-vading grass stem epidermis where there was noobvious evidence of damage. However, thesewere numerically unimportant, and where grassstem epidermis was damaged, much heavier fun-gal colonization was found at the sites ofdamage.

In this investigation, stomata have been foundto be a minor, relatively unimportant place ofphycomycete invasion. Stomatal invasion wascommonly observed only with leaves of grami-naceous plants, and even there direct attach-ment to vascular bundles and mesophyll tissuewas much more common. However, most of theleaves studied were derived from hay or straw,and it is possible that minor epidermal lesions ofthe dried materials may have provided greateraccess to fungal invasion, although lesions werenot obvious.An indication of the extent of plant tissue

invasion by one of these fungi can be seen in Fig.2b. In this case the length of rhizoids extendedto over 450 jim, compared with the sporangiumwhich was only 41 ,im long. A thallus with alarger sporangium may colonize even more planttissue.The time scale of fungal development in the

rumen is also of importance. Although develop-ment of the phycomycetes on rumen plant frag-ments remains to be investigated in detail, theresults from experiments with plant fragmentssuspended in the rumen indicate that the lifecycle covers approximately 24 h, by which timelarge sporangia up to 92 ,um long (Fig. hla andb) were produced. The retention time of plantfragments in the rumen is of a similar order (M.J. Ulyatt, personal communication).The key to understanding the life cycle of the

rumen fungi is the appreciation of the centralrole of plant fragments in zoospore attachmentand fungal development. In the present work ithas been uncommon to find sporangia free instrained rumen contents. This suggests thattheir frequently reported presence there (4, 5, 7,8) may be a direct result of the manipulativeprocedures used in straining the rumen contents.It appears likely that free sporangia found pre-viously, with or without rhizoid fragments at-tached, may have been broken off from the mainportions of the rhizoids, which remained withinthe tissues of the rumen digesta plant fragments.In view of this and the large numbers of funginow shown to be present on rumen plant frag-ments, it is clear that counts of these free spor-angia in strained rumen fluid (4, 5, 7) do not givea true indication of the quantity of fungal tissuein the rumen.

The low numbers of zoospores previouslyfound in strained rumen fluid (4, 7) have alsoappeared to indicate that these fungi are unim-portant in the rumen microbiota. However, therapidity of zoospore attachment to plant frag-ments suspended in the rumen at different timesof day suggests that at any given time the freezoospore count is merely an expression of thezoospores being turned over between sporangialrelease and plant attachment. A primary func-tion of zoospores is to attach to a suitable sub-stratum, and the rapid attachment of high num-bers of zoospores reported in this work indicatesthat they must be constantly replaced to main-tain the zoospore count, albeit low, in rumenfluid. The argument for continuous turnover ofzoospores is further supported by the finding ofdifferent stages of fungal development on thesame plant fragments, a finding most readilyexplained by zoospore colonization at differenttimes during the day.

It is concluded, therefore, that unlike the ru-men bacteria or protozoa, the importance of thefungi cannot be assessed by enumeration of anyof the stages of their life cycle. Zoospores cannotserve this purpose because of their rapid attach-ment to plant fragments. Although counts ofsporangia on plant fragments might be moremeaningful, quantitation would appear to bevery difficult. Therefore, direct measurement offungal mass will be required. It is the quantityof fungal vegetative rhizoid tissues and theirenzymic activities within the tissues of digestaplant fragments that holds the key to assessingthe importance of these fungi in the rumenfermentation. Quantitation of the fungal rhizoidspresent within plant tissues of the rumen willundoubtedly present a problem difficult to re-solve. Nevertheless, the present results alonesuggest that the rumen anaerobic fungi may besignificant members of the rumen microbiota.The extent of attachment, colonization, andgrowth of the fungi on fibrous plant fragmentssuggests also that they may have a role in fiberdigestion in the rumen, perhaps as initial colo-nizers in ligno-cellulose digestion.

ACKNOWLEDGMENTSI thank D. Hopcroft for preparation of samples for SEM

and for assistance with operation of the SEM. I am indebtedto R. T. J. Clarke for discussion.

LITERATURE CITED1. Bauchop, T., and R. T. J. Clarke. 1976. Attachment of

the ciliate Epidinium Crawley to plant fragments in thesheep rumen. Appl. Environ. Microbiol. 32:417-422.

2. Hungate, R. E. 1966. The rumen and its microbes. Aca-demic Press, Inc., New York.

3. Hungate, R. E. 1969. A roll tube method for cultivationof strict anaerobes, p. 117-132. In J. R. Norris and D.W. Ribbons (ed.), Methods in microbiology. AcademicPress, Inc., London.

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4. Orpin, C. G. 1975. Studies on the rumen flagellate, Neo-callimastix frontalis. J. Gen. Microbiol. 91:249-262.

5. Orpin, C. G. 1976. Studies on the rumen flagellate Sphae-romonas communis. J. Gen. Microbiol. 94:270-280.

6. Orpin, C. G. 1977. Invasion of plant tissue in the rumenby the flagellate Neocallimastix frontalis. J. Gen. Mi-crobiol. 98:423-430.

7. Orpin, C. G. 1977. The rumen flagellate Piromonas com-munis: its life-history and invasion of plant material in

the rumen. J. Gen. Microbiol. 99:107-117.8. Orpin, C. G. 1977. The occurrence of chitin in the cell

walls of the rumen organisms Neocallimastix frontalis,Piromonas communis and Sphaeromonas communis.J. Gen. Microbiol. 99:215-218.

9. Orpin, C. G., and L. Bountiff. 1978. Zoospore chemo-taxis in the rumen phycomycete Neocallimastix fron-talis. J. Gen. Microbiol. 104:113-122.

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