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Environmental aspects of Mortierellawolfii infection in cattleP.K.C. Austwick a ba Ruakura Agricultural Research Centre , Ministry of Agriculture andFisheries , P.B., Hamilton , New Zealandb The Zoological Society of London , Nuffield Institute ofComparative Medicine , Regent's Park, NWI 4RY , London , U.K.Published online: 28 Nov 2012.

To cite this article: P.K.C. Austwick (1976) Environmental aspects of Mortierella wolfiiinfection in cattle, New Zealand Journal of Agricultural Research, 19:1, 25-33, DOI:10.1080/00288233.1976.10421042

To link to this article: http://dx.doi.org/10.1080/00288233.1976.10421042

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INTRODUCTIONFrequent detection in New Zealand of

phycomycetes in aborted bovine foetal andmaternal material (Cordes & Shortridge 1968)and the probability that a high proportion ofthese isolates were of Mortierella spp. suggestedthat a search for fungi of this genus in theenvironment of the affected dairy cattle mightreveal the normal substrate of the pathogenicspecies. Just before this study began in 1969the main fungus causing mycotic abortion inNew Zealand had been identified as M. wolfiiB.S. Mehrotra & Baijal, which was at the sametime isolated from the casing soil of a silagestack by di Menna et al. (1972). Subsequentlythe experimental disease has been studied byCordes et al. (1972a, b) .

Mycotic abortion is a major reproductivedisease of cattle in the North Temperate Zone,and the main source of infection is thought to bemouldy hay and straw containing large numbersof spores of the causal fungi, especially

25

Environmental aspects of Mortierella wolfiiinfection in cattle

By P. K. C. AUSTWICK

Ruakura Agricultural Research Centre, Ministry of Agriculture and Fisheries,P.B., Hamilton, New Zealand*

(Received 18 June 1975)

ABSTRACT

Mortierella wolfii B. S. Mehrotra & Baijal, the main cause of mycotic placentitis in NewZealand, was frequently found in over-heated, dark coloured, wet, and rotting plant material ofpH 8-9. Isolates were obtained from 62% of samples of rotting silage, 77% of rotting hay, andfrom soil around silage stacks and pits. No isolates were obtained from good quality silage orhay and very few from visibly mouldy material. The mycoflora of the rotting material wasdominated by thermophilic fungi, including M. ambigua, Petriellidium boydii, Absidia ramosa,Coprinus cinereus, Scytalidium thermophilum, and Stilbella thermophila. Mortierella spp. werenot isolated from the rotting surface layers of an unopened experimental silage stack in whichthe temperature had risen to 68°c during maturation. Material from the outer 2 ern supportedM. wolfii growth in vitro. This suggests that M. wolfii belongs to the secondary successionwhich invades silage after the pit or stack has been opened, or hay after wetting duringfaulty storage. The morphological form in which M. woliii occurs in the field was notdetermined.

Aspergillus [umigatus, Absidia ramosa, andMucor pusillus (Ainsworth & Austwick 1973).Gregory et al. (1963) have extensively docu­mented the occurrence of moulds in hay and instraw, and Lacey (1971) has shown that mouldygrain is almost as good a source of these fungi.A statistical relationship of the incidence ofmycotic abortion during the winter season insouth-east England to the number of days onwhich rain fell in the previous June (i.e., hay­making time) reaffirms the idea that most mouldgrowth in hay occurs immediately after hay­making (Hugh-Jones & Austwick 1967).

In none of these environmental investigations,however, has there been anv mention of theisolation of Mortierella spp. This may be becausethe isolation methods used failed to detect them,as experience has shown that normal dilutionplating methods are unlikely to yield Mortierellaspp. Moreover, the type locality for M. wolfii issoil in India (B. S. Mehrotra & Baijal 1963);the only other occurrence recorded is in soilfrom slag heaps in central England (Evans1971a, b).

* On leave of absence from the Ministry of Agri- Gregory et al. (1963) indicated that theculture, Fisheries a!1d Food, Central Veterinary moisture content of a crop at storage was theLaboratory, Weybrldge,. Surrey, U.K. Pres~nt prime factor determining subsequent developmentaddress: Nuffield Institute of Comparative f h f . I fl . d d f dMedicine The Zoological Society of London 0 t e unga ora m ry-store ee. However,Regent's 'Park, London, NWI 4RY, U.K. 'because many Mortierella spp. prefer very wet

N.Z. Journal of Agricultural Research 19: 25--33

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26 N.Z. JOURNAL OF AGRICULTURAL RESEARCH, VOL. 19. 1976

Fig. 1 - Comparisons of pH and appearance of fieldand experimental grass silage, and the isolation and

growth of Mortierella wolfii on the silage.

beneath the polyethylene in order to record themaximum effect of insolation.

Examination of samplesIn the laboratory pieces were removed from

the centres of samples, to avoid contamination,and placed in 7-cm diameter plastic petri disheswith sterile distilled water added to restoremoisture to 70% if necessary. Plates were placedinside plastic bags which were sealed to preventmites or insects escaping. Incubation was at35°e with a daily examination under X 30 magni­fication for growth, starting at 6 h and continuingfor 15 days. The pH of the samples was alsomeasured. To obtain isolation in pure culture,fruiting heads of Mortierella and other fungi weretouched with a sterile moistened needle and theadhering spores transferred to 2% malt extractagar plates containing 0.01 mg aureomycin and10 mg thiabendazole per ml, followed by furtherincubation at 35°e. To study the fungus flora ofthe material, dilution plates were made in lO-folddilutions, using the above medium but withoutthe thiabendazole, and incubated at 27°e and35°e.

Cut tacc at 15 days

m'I

I

I

Field

Isolations

Mortlerella wollii qro wtb c; II

1ilII--oo;;a:

I

I;;-,~I"1-

0

T ~0

I •. ~

1-"

i gi ~

~o

5 i ~'

" . "

Appearance

\

\\\•II

•II

II

~;.

Auger samples .:l!19wks _

~Fleld-

Experlmental-

~-,~

J.,-,

-, '.III

III

II

p H

,,,•/

//

/

/./II

1T•-('.'

14

'6

'5

11

12

10

13

17

23

24

22

25

27

26

28

52

130

soils with high organic contents (Wolf 1954),it seemed unlikely that over-heating hay or strawwould support the growth of these fungi. Atten­tion was therefore focused on the nextstages of decomposition of hay and straw, aftermoisture contents were raised by leaking roofs,seepage into stacks from the soil, or laterallydriven rain. A parallel investigation was made ofthe mycoflora of grass silage, particularly on thetops and sides of well-matured stacks and pits,where conditions for rotting were comparable tothose in haystacks.

Grass silage microbiology has been largelyrestricted to studying those organisms responsiblefor anaerobic fermentation, especially the lacto­bacilli, and records of the fungal content provideonly a limited picture of the role and speciesof fungi (Hunter 1921). Observations of thephysical and chemical changes in this substratehave been chiefly concerned with the central partsof silage stacks and with the effect of these onthe feeding quality of the product. No recordsseem to exist of the changes effected by matura­tion on the fungal flora of this central part, ofthe external layers beneath the polyethylenecovering, or of the exposed sides of stacks.

MATERIALS AND METHODSTwo limited investigations were carried out.

In one study field samples were collected fromfarms to detect M. wolfii and assess its frequency.In the other, a grass silage stack at RuakuraAnimal Research Station was used primarily toinvestigate the ability of M. wolfii to grow atdifferent sites and stages in the maturation ofsilage. Some observations were also made of theconcurrent mycoflora and of aspects of themicrobial succession in silage.

SamplingField material - Representative samples from

different parts of each silage stack, haystack, orother food material were placed in plastic bagsfor removal to the laboratory.

Experimental material - The experimentalgrass silage was made on 3-4 November 1969 in astack 30 m X 10 m, which was initially 2 mhigh but which sank to 1.5 m as it matured.It was covered with black polyethylene sheetsheld by bands of wire stretched taut by heavytimbers on either side. Core samples from theouter 30 ern were taken 10, 17, 32, 79, and 125days after harvest, at sites on the top and 70 emand 30 ern from the ground on the north side.Temperature in the stack was measured atdifferent depths and, by a continuous remoterecording thermograph, at a point immediately

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AUSTWICK: Mortierella wolfii INFECTION IN CATTLE 27

Small pieces of silage and other materials weremounted in water or in 10% potassiumhydroxide, allowing direct examination. In thehydroxide mountant staining was carried outby treating the mount with a drop of "Parker blue­black Ouink" and leaving it in a moist chamberfor some hours.

To determine pH approximately 25 g (wetweight) of material was suspended in 25 mldeionised water, in a screw top bottle, andallowed to stand at least 2 h (or overnight) atSac before measurement on a Cambridge pHmeter.Growth of M. wolfii on experimental silage

Another experiment was to determine whetherM. wolfii could grow on samples of the experi­mental silage taken at various distances fromthe top and side surfaces. The stock pathogenicstrain (69/5875/1) was grown on 2% maltagar for 2 days at 35°e, and 5-mm squares ofthe growth were cut from the culture margins.These squares were put face down on the surfaceof discs of silage 5 mm thick, taken successivelyfrom the core sample (30 mm diam.) , which werethen placed in petri dishes. The sampling intervalwas increased at 12 em from the surface until afinal depth of 30 em was reached. Incubationwas in plastic bags at 35°e.

RESULTSFIELD SAMPLES

Relationship of pH to origin of samplesand appearance

Types of feed examined are shown in Table 1,and the classification into good, mouldy, androtten relates to their appearance to the nakedeye (Fig. 1). The harvested crops examined,i.e., silage, hay, and straw, have been groupedtogether because the mycoflora of the rottenmaterial was virtually the same. The good haywas sweet-smelling and yellow-green, and thenormal silage, when directly opened in the stack,was olive-green, very moist, and had a charac­teristic acidic smell. The mouldy hay wasgenerally grayish-brown and frequently releasedclouds of A. ramosa spores when handled. Theone pH determination made of this material waspH 7.5. Mouldy silage was rather darker thanthe normal, had little smell, and gave a pH rangeof 6.2-7.8 (average 7.2). Actively growingmycelia were often present, and once the tempera­ture in such an area was 9°e higher than un­touched silage 1 m behind it in the stack.

The rotten silage was always very dark brown,slimy, and dense, and contained the remainsof plant fibres. When dry it was friable and

had frequently been invaded by insect larvae.The pH ranged from 5.5 to 9.8 (average 8.0).The rotten hay was' characteristically very wet,decomposed, dark brown, and with a pH of5.5-9.7 (average 8.0). Samples were mostlytaken from the bases of stacks or from the sideswhere rain had been driven into the exposedhay or had leaked from faulty roofing.

Determinations of pH on the cut faces of twosilage stacks showed very rapid change onexposure to air, assuming the original level wasabout pH 4.5. In one stack the face had beenexposed for 3 days, and the top decomposedlayers were at pH 9.8. The layers 7-8 emfrom the top were at pH 9.3, and at a depthof 23-25 em the pH was 8.5 in the presenceof pronounced bacterial decomposition and manyfly larvae (Musca domestica). The other stackhad been open for 3 weeks and showed extensivedecomposition where water had run down fromthe top. In the centre of the cut surface, at adepth of 7 em, the pH was 8.4 (Fig. 1).Mycoflora

Table 1 lists the fungi recovered from thesamples and shows, even with the relativelysmall number of records, that some species wereobtained only from rotting material whereasothers never occurred on this type of substrate.A few species, such as Petriellidium boydii, wereobtained from all substrates sampled. Thequantitative aspect relates only to the numbersof samples from which the species was recovered.

The main distribution of rotten silage was ina thin layer over the top of the stacks or pits andin a thicker layer over the sides, but rotten hayoccurred anywhere in the stacks where sufficientwater had penetrated.

(1) Mortierella spp.These were detected in feed on all but one

of the farms on which at least one mycoticabortion had occurred. Mortierella spp. wereisolated from rotten hay on six of the seven farmswhere hay was being fed and from rotten silage onall eight where this was in use (Fig. 3). Isolateswere also obtained from two of the five unaffectedfarms where silage was fed, but not from anysample of good hay, silage, or grass.

The commonest habitat of Mortierella spp. wasrotten material, and they were found in 77.0% ofthe rotten hay and 62.1% of the rotten silagesamples. The positions in a stack from whichM. wolfii was recovered seemed to depend onthe amount of rotting which had occurred. How­ever, one face only 3 days after cutting gaveisolates at 8-9 em from the surface, in an areajust beginning to rot but with a pH range from

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28 N.Z. JOURNAL OF AGRICULTURAL RESEARCH, YOL. 19, 1976

TABLE I - Feedstuffs, etc., examined and fungi isolated

No. samples examined SilageHayStraw

Rotten

3113

I

Mouldy

63

Good

151

Soil Grass

Totals 45

No. of premises from which samples were 21examined

Species observed(No. premises from which obtained)

Absidia butleri Lendner ItAbsidia ramosa (Lindt) Lendner 8Mortierella ambigua B.S. Mehrotra 5Mortierella wolfii B.S. Mehrotra & Baijal IIMortierella spp. indet. 6Mucor pusillus Lindt 3

tRhizopus sp, ItChaetomium anguipillum L. Ames 5tMicroascus cirrhosus Curzi 2Monascus purpureus Went -

tPetridiellium boydii (Shear) Malloch 17Saccobolus saccoboloides Seaver apud I

Dodge & SeavertSordaria humana (Fuck.) Wint.Thielavia fimeti (Zop£) Malloch & Cain IAspergillus candidus Link I

tAspergillus [umigatus Fres. 5Aspergillus glaucus series I

tAspergillus nidulans Eidam 2Aspergillus terreus Thorn IAspergillus versicolor (Vuill.)

TiraboschiCandida albicans (Robin) BerkhoutCandida krusei (Castellani) Berkhout

tCephalosporium spp. 9Cladosporium sp. I

tEpicoccum nigrum Link 2tFusarium spp. 5Geotrichum candidum Link -Humicola sp, 3

tMalbranchea pulchella var. sulphurea 5Sacco et Penz

tPenicillium sp. IPhoma spp, IRhodotorula sp. IScopulariopsis brevicaulis (Sacc.) 2

Bain.tScopulariopsis sp, 2

Scvtalidium thermophilum (Cooney & 2Emerson) Austwick

tStilbelia thermophlla Fergus 4Stvsanus spp. 2Thermomyces lanuginosus Tsiklinsky IThermomyces stellatus (Bunce) Apinis ITrichothecium roseum Link 4\'erticillium sp.

tCoprinus cinereus (Schaeff. ex Fr.)S.F. Gray 4

9

8

3

2I

73

3

2I

16

13

2

I43

2

2

2I

3

6

6

I2II

2

2

2

2

tobtained from experimental silage

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AUSTWICK: Mortierella wolfii INFECTION IN CATTLE 29

6.4 to 9.3 (see Fig. 1). Oiher isolates wereobtained from mouldy silage, from soil adjacentto silage stacks, and from rotting straw. Mouldyhay did not give Mortierella spp. and neither didany of the hay and silage samples of normalappearance.

Most isolates identified were M. wolfii, a fewwere M. ambigua, and five other morphologicaltypes were noted. M. wolfii and M. ambiguagrew well at 35°c, the temperature consequentlyused for all isolations. Mortierella spp. wererecovered from 78 of 354 (22%) dilution platingsof feed samples; from the rotten hay and silagethe recovery rate was 29.5% (70/237 platings).This indicates the difficulty of isolating thesefungi by dilution plating compared with the directincubation of samples, even though thiabendazolecan be used to inhibit non-phycomycetous moulds.Quantitative determinations of Mortierella bythis method were therefore precluded. Therecorded recoveries from rotten hay and straware also probably of little use in this respect,because they are calculated from samples fromall parts and depths of different stacks.

(2) Other fungiConcentration on the probable habitats of

M. wolfii has given a biased view of the mycfloraof the material examined, e.g., most isolations werecarried out at 35°c so that many of the fungiobtained are thermophilic species, most of thempreviously known from mouldy hay and compost.

Petriellidium boydii (Shear) Malloch (syn.Allescheria boydii Shear, sta. conid. Monosporiumapiospermum Saccardo) was the commonestspecies observed. It varied in appearance accord­ing to the relative abundance of conidia,ehlamydsospores, coremia, or c1eistothecia. Nopathogenicity tests were carried out. P. boydii isassociated with bovine abortion overseas, so it issurprising that no cases have been reported inNew Zealand. From its appearance in pasturegrass and good silage samples when incubated at35°c P. boydii is assumed to be one of the mainhigh-temperature decomposers of organic matter.However, it was recovered from the experimentalsilage only from day 32 onwards, which mayindicate that a substrate pre-conditioning wasnecessary for development.

Absidia ramosa (Lindt) Lendner is a commonsaprophyte which seems to be more frequent onNew Zealand substrates than on comparable onesin Britain, most notably on mouldy hay where itwas often the dominant organism. The relativeinfrequency of Aspergillus [umigatus in bothmouldy hay and in mycotic abortion was one ofthe major differences noted.

r-ig. 'L - Coprinus cinereus fruiting beneath polye­thylene cover on stack of experimental grass silage.

Scytalidium thermophilum (Cooney & Emer­son) nov. comb. (basinym. Torula thermophi/aCooney & Emerson (1964) is a common thermo­philic species which forms long chains of dark­coloured thallospores (up to 18 p. diam.). Thisfungus clearly belongs to the genus ScytalidiumPesante in the sense of M. B. Ellis (1971), towhich it is now formally transferred.

Sti/bella thermophila Fergus is a characteristicmember of the rotting silage community andgrows readily at 35°c at the end of the succession.It was first isolated from mushroom compostin Switzerland by Fergus (1964).

Coprinus cinereus (SchaefI. ex Fr.) S. F. Gray(syn. C. macrorhizus (Pers. ex Fr.) Rea,C. fimetarius (L. Fr.) appeared in five culturesof field material and also grew profusely alongthe sides of the experimental stack beneath thepolyethylene cover some 19 weeks after silagemaking . The species is a characteristic dung­inhabiting form (Fig. 2) .

Other species in Table 1 were sporadic intheir occurrence. Thus, it appears that the con­ditioning of substrates favoured by Mortierellaspp. is not suitable for the survival of a great

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30 NZ. JOURNAL OF AGRICULTURAL RESEARCH, VOL. 19, 1976

TABLE 2 - Experimental silage: pH of samples fromouter and inner zones of stack

Samples taken Outer zone Inner zoneDepth pH Depth pH(ern) (em)

November 1969 1.0 8.0 28.0 4.51.0 9.1 27.0 4.5

January 1970 2.0 8.6 18-20 5.21.0 8.7 19-20 5.1

March 1970 0.1 8.8 27-28 4.8

Mean 8.64 4.82

many moulds, even after they have played a rolein the earlier succession such as in the mouldingof hay. Not many common saprophytic speciesdevelop in mouldy and rotting silage, and thefungal succession which starts when the stack isopened is quite different from the primary oneof mouldy hay.

EXPERIMENTAL SAMPLES

Appearance, temperature, and .pH ofsamples

Apart from the maximum temperature reachedduring maturation, the main finding of interestwas the diurnal variation in the partially rottedouter layers of the stack during the hottest periodsof the summer. The highest temperature recordedduring maturation was 68°c on day 17, at a depthof 30 em and 90 em from the ground. At thesame time the temperature at 25 em beneaththe top surface was 55°c. Later, the internalstack temperature stabilised at 35°c.

During 28 days of running between December1969 and February 1970, the continuous­recording, distant-reading thermometer placedimmediately beneath the polyethylene cover gavean average daily maximum of 43.2°c and anaverage minimum of 19.6°c. The maximumtemperature reached was 51.5°c, and 47.0°c wasexceeded on 8 days, which is well over the limitfor active growth for most micro-organisms.

The close connection between gross appearanceand pH of the silage was similar to that seen inthe field samples (Table 2). The inner silage(anaerobic) was a normal, light olive-brown withan acid smell and an average pH of 4.82 (fivesamples taken 20 em from top surface). Theouter layer was dark brown, very moist, anddecomposed at the top but retained much of itsstructure below 2.0 emfrom the surface. The top1.0 cm had an average pH of 8.64 (five samples)and had only a slightly fetid smell when mature(Fig. 3).

Samples from the side of the stack were similarin appearance and pH value except that the earlydecomposition spread much further into thesilage than at the top. At each site the lastsampling, at 19 weks in March 1970, showedthat the thickness of decomposed silage was abouthalf that found in the first sample, probablybecause of the evaporation and compressionwhich occurs during the first few months. Thedry hot summer of 1969-70 no doubt contributedto this. Fig. 3 graphs the appearance of thesilage in relation to pH, position in the stack,and age.

MycoftoraAlthough Mortierella spp. were not isolated

from any of the samples of experimentally madesilage, other fungi listed under (t) in Table 1were obtained, occasionally abundantly, in bothdirect cultures and dilution plates (Fig. 3). Themain species was P. boydii, isolated from threeout of the five samples.

The chief distinction between the isolates wasin their distribution in the decomposed andundecomposed parts of the stack (Table 1). Ofthe 20 species recorded in 5 series of samples,9 occurred only in the decomposed and 5 onlyin the undecomposed parts, indicating a fairlyspecific flora for each part. Overall recovery wasrelatively low, for with a possible 100 species persample opportunities for isolation, only 28recoveries were made in the decomposed silageand 12 in the good quality silage below it. Fungi,notably P. boydii, were isolated from a depthof 10-18 ern in the first 2 weeks at the two siteson the side of the stack (Fig. 3) , but no recoverieswere made deeper than 7 em thereafter. Nofungi were ever isolated deeper than 2 em fromthe top surface.

Although the number of samples was small, itindicated that well-prepared and matured grasssilage is a relatively poor substrate for fungicompared with moist hay, probably because of therestricted aeration, but that it does have acharacteristic fungal flora. There is also someevidence that whereas the recoveries from theundecomposed silage fall off as the stack ages,the numbers of species possibly increase withinthe decomposed layers (Table 3).

Growth of Mortierella wolfii onexper-imental silage

In the growth trials on experimental silage,samples taken at 3 weks from the top 0-1 emfailed to support the growth of M. wolfii, despitetheir decomposed state and high pH (Fig. 1) . Onesample taken at 3 weeks from 2.5-3.0 em belowthe surface allowed growth of the inoculum for a

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AUSTWICK: Mortierella wolfii INFECTION IN CATTLE 31

Key,

a Side 30 emb Side 90 eme Top 0 em

PRESENCE OF

Key,

;~~Dark brown __ ~

Dark olive -green _ -

pH

4'0 5'0 6'0 7'0 8'0 9·0

)!'

'" .\

'"2 '""3 /..~_.-.... , .,4 '/

"I5

"I

6,.,I...

7 II

8..,I'

E i:o 9~

1"r.I 10 I,.....~ ."a. 11w II·0 r:12

"I.,13 I"

Ii;14 Ii;15 'i'I'16 .r17 I Key,

I Top- 2wks -18I 19wks --

19 ,Side -30em 19wks -.-90em 19wks .....

f .g. 3 - Experimental grass silage: changes during maturation in pH, appearance, andpresence of fungi.

TABLE 3 - Number of fungal species per sampling

Weeks after harvest1112 2112 4112 11 19

short time. No growth was obtained on samplesfrom lower levels. At 11 weeks only the top0-1 em supported growth, and the inocula onsamples down to 28 em died out. At 19 weeksit was still possible to obtain growth only onsamples down to 2 em.

The side samples were rotted to a much greaterdepth, and one taken 3 weeks after harvest at90 em from the ground supported the growth ofM. wolfii to a depth of 14-15 em. Samples takenat the same site at 11 weeks supported growthonly to a depth of 5-6 em, but those at 14 weeksshowed growth at 20 em deep. At 10 weeks, how-

Decomposed silage 2Undecomposed silage 5

62

2

492

51

ever, no growth was made on any sample. Noneof the side samples at 30 em from the groundsupported growth when they came from positionsdeeper than 3-4 em, and, although the decomposi­tion was still pronounced, the pH was sur­prisingly low (c. 5.0).

DISCUSSIONThe present investigation clearly indicates that

Mortierella spp., particularly M. wolfii, are verymuch organisms of wet substrates with high pHvalues, because no growth of these fungi wasfound in any substrate below pH 7.0. In naturehigh pH values are generally the result ofbacterial anaerobic fermentation of plant material,which may produce free ammonia and otheramines. When coupled with the high temperaturesencountered in both the outer layers of the sidesof the experimental silage stack (up to 68°e)and beneath the black polyethylene cover (up toSlOe), high pH seems to have a highly selectiveaction on the mycoflora, eliminating all but a fewspecies which have heat-resistant spores.

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32 N.Z. JOURNAL OF AGRICULTURAL RESEARCH, VOL. 19, 1976

Attempts were made to determine the sourceof M. wolfii growing in rotten silage, but this wasnot clarified. As no zygospores are known, it isfairly certain that most growth initially occursfrom thick-walled chlamydospores which may giverise either to hyphae or (possibly more com­monly) directly to sporangia. M. wolfii wasapparently absent from the unopened experi­mental stack, so its presence in samples takenfrom the face of a pit opened 15 days previouslycan most probably be explained by very rapidmycelial spread from the sides of the pit, or byrain splash or wash of spores from the deliquesc­ing sporangia in the rotten silage above.Sporangiospores could possibly be carried on thefeet of flies whose larvae seem to play animportant role in the rapid decomposition ofrecently uncovered silage. One of the character­istics of M. wolfii found by Evans (1971b) is itsability to grow at up to 48°c, which wouldcertainly give it an advantage over other fungiwhen invading such a high-temperature substrate.

M. wolfii would therefore seem to play no partin the early microbial succession of silage matura­tion, entering only as a secondary organism. Notall silage is well-sealed, and this secondary suc­cession will commence wherever oxygenpenetrates, probably progressing inwards thelonger the silage is kept. In this way the outer,rotten, weathered silage may provide a concentra­tion of M. wolfii propagules in an infective form,i.e., supposedly sporangiospores or chlamydo­spores, and it is this part of the silage that iseaten first by cattle (preferentially, according toSOIne farmers).

Some evidence of the consequences of silageinvasion by M. wolfii was shown in the two dryseasons of 1967-68 and 1969-70. Silage had tobe fed as a supplement very early, and M. wolfiiabortion started some 2 months earlier than inJ968-69 (Carter et al. 1973), when grass grewthroughout the summer (Austwick & Cordes,unpublished observations) .

The amount of M. woljii in the .rotten silagecould not be assessed, for the reasons discussedabove, but such an estimate could prove vital toadvising a farmer of the potential danger infeeding a particular batch of silage. As a resultof this study, the general recommendation can bemade that rotten silage from the top and sides ofpits and stacks should certainly not be fed topregnant cattle and is probably best destroyed.Also, precautions should be taken to minimisethe time that a cut face of silage is left exposedbefore the next cut is made.

So little is known about the mycoflora of silagethat New Zealand grass silage cannot be com­pared with silage made from maize, alfalfa,sorghum, or other crops. There is certainly acharacteristic thermophilic mycoflora, which cor­responds partly with that seen in mouldy hay andstraw but more especially with that observed inmushroom compost. The virtual absence of yeastsemphasises the difference between grass silage andthe wet-stored grains now commonly used forstock feeding.

di Menna et al. (1972) considered that pre­pagules of M. wolfii were not frequent in thefarm environment but qualified this statement byemphasising the difficulties of isolation by dilutiontechniques in the presence of other mucoraceousmoulds. The present study shows that adoptionof a direct incubation technique greatly increasesthe chances of finding M. wolfii. Although it isone of the commoner species concerned in thedecomposition of grass silage, M. wolfii is mainlyrestricted to the specialised habitat of the outerlayers already rotted by bacteria and having ahigh pH.

Acknowledgments

The New Zealand National Research Councilfor awarding a Senior Research Fellowship; andthe Director-General of the New Zealand Ministry ofAgriculture and Fisheries and the Director of theCentral Veterinary Laboratory, Ministry of AgricultureFisheries and Food, Weybridge, for facilitating myvisit to New Zealand" Dr D. G. Edgar, Director ofthe Animal Research Station, Ruakura AgriculturalResearch Centre, Hamilton; Dr J. N. Parle, Mr D. O.Cordes, and their staffs for much help and encourage­ment; and many Waikato veterinarians and farmersfor their kind collaboration. Mr D. McQueen andMr D. Taylor for processing photographs and Mr E.Coleman for preparing the figures.

REF'ERENCESAinsworth, G. C.; Austwick, P. K. C.; 1973: "Fungal

Diseases of Animals". 2nd edition. Review SeriesNo.6. Commonwealth Bureau of Animal Health.Commonwealth Agricultural Bureaux, FarnhamRoyal.

Carter, M. E.; Cordes, D.O.; di Menna, M .E.;Hunter, R. 1973: Fungi isolated from bovinemycotic abortion and pneumonia with specialreefrence to Mortierella wolfii. Research inVeterinary Science 14: 201-6.

Cooney, D. G.; Emerson, R. 1964: "ThermophilicFungi". Freeman, San Francisco.

Cordes, D.O.: di Menna, M. E.; Carter, M. E. 1972a:Mycotic pneumonia and placentitis caused byMortierella woliii. I. Experimental infectionsin cattle and sheep. Veterinary Pathology 9:131-41.

Cordes, D.O.; Carter, M. E.; di Menna, M. E. 1972b:Mycotic pneumonia and placentitis caused byMortierella wolfii. 11. Pathology of experimentalinfection of cattle. Ibid. 9: 190-201.

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AUSTWICK: Mortierella wolfii INFECTION IN CATTLE 33

Cordes, D.O.; Shortridge, E. M. 1968: Systemicphycomycosis and aspergillosis of cattle. NewZealand Veterinary Journal 16: 65-80.

Ellis, M. B.; 1971: "Dematiaceous Hyphomycetes",Commonwealth Mycological Institute, Kew.

Evans, H. C. 1971a: Thermophilous fungi of coalspoil tips. I. Taxonomy. Transactions of theBritish Mycological Society 57: 241-54.

1971b: Thermophilous fungi of coalspoil tips. II. Occurrence, distribution andtemperature relationships. Ibid. 57: 255-66.

Fergus, C. L. 1964: Thermophilic and thermotolerantmoulds and actinomycetes of mushroom compostduring peak heating. Mycologia 56: 267-84.

Gregory, P. H.; Lacey, M. E.; Festenstein, G. N.;Skinner, F. A. 1963: Microbial and biochemicalchanges during the moulding of hay. Journal ofGeneral Microbiology 33: 146-74.

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eli Menna, M. E.; Carter, M. E.; Cordes, D. O. 1972:Identification of Mortierella wolfii from cases ofabortion and pneumonia in cattle and a searchfor its infection source. Research in VeterinaryScience 13: 439-42.

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