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Fungal colonization of rice straw and palm press fibre in the rumen of cattle and buffalo
Fungal colonization of rice straw and palm press fibre in the rumen of cattle and buffalo
ABSTRACT Ho, Y.W., Abdullah, N. and Jalaludin, S., 1991. Fungal colonizstionofricestraw and palm pressfibre in the rumen of cattle and buffalo. Awn. FeedScr. Technol.. 34: 3 l-321.
I
Fungal colonization and development in rice straw and palm pressfibre (mesacarp ofoil palm fruit after the extraction ofoil) were very similar in both cattle and buffalo. In both animal species,atlachmeat to rice straw by rumen fungal zoosporeswas rapid, within 15 min of umen incubation. At 6 h of mmen incubation, thin- and thick-walled tussles were colonized by fun@ hyphae and by 24 h fungal calonization was exIensive. Fungi were predominamlv thosewith spherical and ovoid smmmgia. Fusiform sporangia were in fcwcr numb&. Fungal colo&ation was also extensive at 48 and 72 h. The colonization and development ofthe fungi on the palm presstibre, both untreated and treated wilh ammonium hydroxide and ether. was slow.
INTRODUCTION
Studies on the anaerobic rumen fungi in the past 15 years have indicated that they are important colonizers of plant tibres (Orpin, 1977a, b, 1981; Bauchop, 1979a, b, 1981, 1989; Akin et al., 1983; Akin and Rigsby, 1987). They show a preference for the thick-walled lignitied cells, such as the sclerenchyma cells and vascular elements (Akin et al., 1983; Akin and Rigsby, 1987; Ho et al., 1988a) and have been reported to contributeconsiderably to the overall digestion of various forages and wheat straw (Akin et al., 1983; Gordon and Ashes, 1984; Akin and Rigsby, 1987). Low-quality roughages, such as fibrous crop residues, have in recent years been studied as a potential feed resource for ruminant animals in the tropics. However, knowledge on the digestion of these fibrous crop residues by rumen microbes, particularly the fungi, is lacking. The present study was carried out to examine the invasion and colonization by rumen fungi on two tibrous crop
0377-8401/91
I$0350
0 1991 Elsevier Science Publishers B.V. All rights reserved.
Y.W. HO ET AL.
3i?
residues, viz. rice straw and palm press tibre (mesocarp tibres of oil palm fruits after the extraction of oil), which are being studied as potential feed materials for cattle and buffaloes. MATERIALS
AND METHOCS
Two Kedah-Kelantan cattle (80s in&us) and two swamp buffaloes (Bubalus bubalis), approximately 18 months old, and all males, each with a permanent fistula in the rumen, were maintained in separate cages indoors. Each animal was fed once daily (O&30 h) with guinea grass (Panicum maximum) ad libitum and each had free access to mineral blocks (cobalt mineralized salt blocks) and water. Fibrous crop residues (rice strawand palm pressfibre) Rice straw and palm press fibre (PPF) were dried and ground through a 4 mm screen in a hammer mill. The rice straw used was untreated, but PPF, both untreated and treated with ether (for fat extraction) and ammonium hydroxide (NH&H) (for softening of fibres), was used. PPF was subjected to fat extraction (2-3 g per thimble) by refluxing with petroleum eiher (60-80°C) in a Soxhlet condenser reflux for 8 h and dried at 65°C for 48 h before use. The PPF contained 4% ether extract. For NH,OH treatment, PPF was incubated for 8 days with 3% NH,OH (v/v) at ambient temperature (Vijchulata et al., 1985). The above treatments of PPF were carried out to determine whether the removal of fat and softening of the fibres would improve colonization of the fibres by fungi. Chemical analyses of straw and PPF Chemical analyses for dry matter (DM), lignin and nitrogen were conducted according to standard procedures (Association of Official Analytical Chemists, 1980). Neutral detergent fibre (NDF) was determined following the method described by Goering and Van Soest ( 1970). Table 1 shows the chemical composition of straw and PPF, while Table 2 shows the in situ digestibility of straw, untreated PPF, ether-extracted PPF and ammonia-treated PPF determined by the nylon bag technique (Orskov et al., 1980). Incubation ofrice straw and PPF in the rumen About 1 g of material was placed separately in nylon bags (90x60 mm, mesh size SOpm). The bags were tied in the same manner as described by Ho
Chemical composition of straw and PPF Comoosition 1%)
SIIZW
PPF’
DM Crude protem NDF Lignin Ether extract
86.0 4.3 73.0 19.0 1.3
70.6 6.2 77.6 21.3 4.0
‘From previous studies on amm.xGa treatment of PPF. the chemical composition of the tihre listed above was not significantly changedby the treatment. TABLE 2 Percent DM loss at 48 h of rumen incubation of straw, and untreated and treated PPF Feed
Cattle
Buffalo
straw Untreated PPF Ether-extracted PPF Ammonia-treated PPF
49.3k5.6’ 20.7fO.7” 17.3kO.8” 35.s*o.7”
53.2i 1.4b 2i.6f0.2’ 19.6kO.6” 39.9+0.5b’
Means with different superscriptsin the same row differ significantly (PcO.05 ). ‘Jalaludin et at. (1984 ). et al. ( 19%) and were placed in the ventral section of the tumen. The bags with rice straw were incubated for I5 and 30 min, 1, 3,6, 24,48 and 72 h, and those with PPF for 6,24,48 and 72 h. After retrieval, the bags were washed with water and the samples fixed in appropriate solutions.
Preparation of samples for scanning electron and light microscopy Samples for scanning electron microscopy (SEM) and light microscopy observation were. prepared using the method described by Ho et al. (1988b). For every incubation period of each material in each animal, 15 fragments were studied with SEM and 20 fragments were observed with light microscopy. RESULTS AND DISCUSSION
Invasion and colonization of rice straw and PPF, untreated and treated with NH,OH and ether, by rumen fungi appeared similar in both cattle and buffalo. Table 3 shows the prevalence of fungal sporangia on each material at various incubation times En both species of animals. Rice straw fragments were observed to have more sporangia than PPF at the various incubation
Y.W.HO ET AL.
314 TABLE 3 Prevalenceof funpa! sporangia on rice straw and PPF Animal/malerials
lncubalion lime I5min
30min
Ih
3h
6h
24h
48 1
72h
Cattle
straw
+
t
+
+
++
+++
_
_
-
-
+tt
PPF PPF-E PPF-A
+ + +
+ + +
ct ++ ++
++ ++ ++ t+
+
+
+ -
+ _ _ -
++ + + +
+++ + + +
++t ++ ++ +t
++ tc +t t+
BUffal straw PPF PPF-E PPF-A
_
f, few sporangia; ++. moderate number of sporangia; +++, determmed. PPF-E, ether-extracted palm presslibre. PPF-A, ammonia-treated palm pressfiba.
abundant sporangia; -,
not
periods. The occurrence of sporangia on the treated and untreated PPF appeared to be very similar. Fungal zoospores were found to attach on rice straw fragments 15 min after rumen incubation and by 30 min many had germinated. Similar rapid attachment of zoospores to plant fragments in the rumen has been reported by Bauchop (1981) and Hc et al. (1988a). The zoospores attached predominantly on stomata, cracks, fissures and cm suifaces of the rice straw frag ments. Similar sites for attachment have been observed by other workers (Orpin, 1977b; Bauchop, 1979b; Akin et al., 1983; Ho et al., 1988a). By 1-3 h, some fungi had developed an extensive hyphal system in the thinwalled parenchyma cells of the rice straw. Some of the hyphae produced lobed or disc-shaped ‘appressoria’ with penetration pegs penetrating cell walls. Fungi with ‘appressoria’ colonizing guinea grass in the rumen of cattle and buffalo have been reported earlier by Ho et al. (1988a). After 6 h in the rumen, the thick-walled lignified tissues, such as sclerenchyma and vascular elements, were colonized by the fungi. Degradation of the cell wall occurred in the thinwalled tissues which were extensively colonized by fungi. Some degradation was also observed in the thick-walled tissues with profuse fungal colonization. Sporangia of the fungi were small, 14-18x 14-20 pm, mostly spherical or ovoid. At 24 h, fungal colonization of the straw fragments was very profuse (Fig. 1). Dense networks of hyphae were observed in the sclerenchyma and vascular tissues. Vascular cylinders were easily detached because of degraded tis-
315
Fig. I. (A) Fungal colonization of rice straw 24 h after incubation in the rumen of caltk. Bar. 30 pm. (B) An uncolonized rice straw fragment showir.g surface struci~~~; PH, prickle hair; PP, papillae; SC, silica crystals. Bar, 50 /urn. sues surrounding
them. Most of the fungal sporangia were much larger (40-150 pm diameter) than those observed at 6 h, and they were mostly spherical and ovoid. Fusiform and ellipsoidal sporangia were also present.
but in fewer numbers. Among the fungi which produced spherical or ovoid sporangia, some were polycentric species as several sporangia developed from a single hypha. Polycentric anaerobic rumen fungi have recently been isolated from cattle and buffalo fed a high-roughage diet in various parts of the world (Akin and Rigsby, 1987; Phillips, 1988; Barr et al., 1989; Ho et al., 1990). Fungi with fusiform sporangia were also recently reported to be polycentric species (Ho et al., 1990). By 48 h, much of the softer thin-walled tissues of the rice straw fragments were degraded, and the remaining sclerenchyma and vascular tissues were extensively colonized by fungi. Fungi with spherical or ovoid sporangia still predominated. However, large spherical and ovoid sporangia, like those present at 24 h, were much less numerous. Instead, a large number of smaller ovoid structures (8- 15 pm diameter) which could be germinated zoospores or young sporangia were present both outside and within the cells (Fig. 2). These young sporangia/germinated zoospores did not produce extensive networks of hyphae. Most of the hyphae produced were thick and short. Some of the larger sporangia appeared shrunken or collapsed with part of the sporangial walls disintegrater’ or with openings or pores in the walls (Fig. 3). These collapsed sporangia were similar to those observed in guinea grass by Ho et al. ( 1988a) in the rumen of cattle and swamp buffalo, and they are probably the remains of sporangial wall after the release of zoospores. Warty spores, which appeared similar to those reported by Ho et al. (1988a), were also
Fig. 2. Rice straw fragment 48 h after incubatiat in the rumqof buffalo wall ard small ovoid sporangia in the exposed cell lumina. Bar, 10 #tn.
showing
degraded cell
317
Fig. 3. Remains ofsporangia, after the liberation of zoospores, on a rice stmw fragment 48 h afterrumen incubation in cattle. Bar, 20~m.
observed in a few fragments. These warty spores, as suggested by Ho et al. ( 1988a), may be some kind of resting spores. Fungi with fusiform and ellipsoidal sporangia were more abundant at 48 h than at 24 h, but their numbers were still fewer than those with spherical or ovoid sporangia. These fungi were attached mainly to the vascular cylinders and their hyphae, often with constrictions, were seen colonizing the sclerenchyma and vascular tissues. Although the degradation of tissues was quite extensive at 72 h after rumen incubation, extensive fungal growth was not observed. The number of sporangia was very much less than that at 24 and 48 h. Sections of unincubated PPF showed that each strand of the PPF is made up of thick-walled, highly lignified sclerenchyma cells enclosing vascular tissues in the centre. Longitudinal files of silica cells with clusters of silica lie adjacent to the outermost layer of sclerenchyma cells. Where the silica cells are numerous, they form almost complete sheaths around the PPF strands. Treatment of PPF with ether or NH,OH did not seem to dislodge many of the silica clusters. Invasion and colonization by the rumen fungi on PPF untreated and treated with ether and with NH,OH appeared similar. The untreated PPF was colonized in the same manner as that treated with NH,OH or ether. Grenet and Barry ( 1988) also found that there was no difference in the colonization of rumen fungi on wheat straw untreated or treated with ammonia. Wuliji and McManus ( 1988) reported that pretreatment of sugar cane bagasse, oat straw and wheat straw with ammonia did not increase fungal colonization signifi-
318
Y.W.HO ET AL.
cantly, but for so;aum hay, oat hull and lucerne stem, fungal colonization was improved significantly by the pretreatment. At 6 h ofrumen incubation, fungal colonization on the PPF fragments was very limited. Germinated zoospores and small sporangia were observed on cut ends of fragments, damaged surfaces and cavities left by dislodged silica clusters (Fig. 4). Sporangia were mainly spherical and ovoid. Cylindrical and tiliform sporangia were in lower numbers. Bv 24 h, some fungal hyphae were observed in the lumina of the tibres, particularly near the cut ends. Some of the hyphae produced lobed or disclike ‘appressoria’. Sporangia produced by the fungi were variable in size and they were mostly spherical, ovoid, cylindrical and fusiform. The fusiform sporangia were large (45-130 longx 1S-55 pm wide), usua!ly with long sporangiophores (up to 140 pm long) raising the sporangia above the fragments (Fig. 5). At 48 and 72 h of rumen incubation, fungal hyphae were abundant in the lumina of the tibres. In areas with very profuse colonization by the fungi, some degradation was obsrrved in the cell wall of the fibres. The predominant fungi observed on the surface of the libres were those which produced fusiform sporangia. Small spherical or oval sporangia were observed mainly in cracks and crevices of the tibres (Fig. 6). The results of the present work showed that fungal colonization and devel-
Fig. 4. Germinated zoospore (2) in a cavity (C) left by dislodged silica clusten of a PPF, 6 h after ~umcn incubntion in cattle. S. !.ilica clusters. Bar, 5 pt.
319
Fig. 5. A PPF showing a large fusiform sporangium with a long sporangiophore raising the sgorangium above the fibre, 24 h after rumen incubation in buffalo. Note the limited degradation of tissues. Bar, 20 pm.
Fig. 6. Small spherical or oval sporangia in cracks and crevices of the PPF, 48 h after rumen incubation in cattle. Note the top portion of the fibre which shows some silica cty~Ials and empty cavities left by dislodged crystals. Bar, 20~m.
320
Y.W.HOETAL
opment seemed to be more abundant and rapid in the rice straw than in PPF (both untreated and treated with NH,OH and ether). Fungal colonization and development in both untreated and treated PPF was slow. Although there were significant differences between untreated PPF and PPF treated with NH,OH in percentage DM loss (Table 2), differences in the colonization and development of fungi in these untreated and treated tibres, as observed by SEM and light microscopy in this study, were not apparent. ACKNOWLEDGEMENTS
We thank Professor A. Nawawi, Department of Botany, University of Malaya, for his helpful suggestions, and Kasmawati Mahmood and Suleka Madhavan for their technical assistance and co-operation.
REFERENCES Akin, D.E. and Rigsby, L.L., 1987. Mixed fungal populations and lignoceilulos~c lissue degradation in the bovine rumen. Appl. Environ. Microbial., 53: 1987-l 995. Akin. D.E., Gordon, G.L.R. and Hogan, J.P., 1983. Rumen bacterial and fungal degradation of Digiraria pmr~ii grown wilb or without sulfur. Appl. Environ. Microbic& 46: 738-748. Association of Oflicial Analytical Chemists. 1980. Oflicial Methods of Analysis. 13th Edn. A.O.A.C.. Washington,DC.,:*p. 14-125. Barr, D.J.S.. Kudo, H., Jakober, K.D. and Cheng. K.-J., 1989. Morphologyanddcvclopmcntof rumen funei: N~.?crr//ifl7us~i.rSD.. Pirumvces corntnrrrris. and Orurno~~wccs6ovis ecn. new.. sp. no”. Can. r. 601.. 67: 281 S-2824. Bauchop. T., 1979a. Rumen anaerobic fungi ofcattle and sheep. Appl. Environ. Microbial.. 38: 148-158. Bauchop, T., I979b. The rumen anaerobic fungi: colonizers of plant libre. Ann. Rech. Vet., IO: 246-248. Bauchop. T.. 1981. The anaerobic fungi in lumen libre digestion. Agric. Environ., 6: 339-348. Beuchop. T.. 1989. Biology ofgut an&bic fungi. BioSys&ms, 23: 53-64. Gocrine, H.K. and van Soest, P.J., 1970. Forage Flbre Analyses. U.S. Dept. Agric. Agriculture HandbookNo. 379. Gordon. G.L.R. and Ashes, J.R.. 1984. In vitro digestion of wheat straw by different rumen anaerobic funai. Can. J. Anim. Sci.. 64: 156-l 57. Grcnct, E. and Birry, P., 1988. Colonization of thick-walled plant tissues by anaerobic fungi. Anim. Feed Sci. Technol.. 19: 25-31. Ho. Y.W., Abdullab, N. and Jalaludin, S.. l988a. Colonization of guinea grass by anaerobic rumen fungi in swamp buffalo and cattle. Amm. Feed Sci. Technol., 22: I6 I-171. Ho. Y.W., Abdullah. N. and Jalaludin, S.. 1988b. Peneiratine. structure of anaerobic mmen fungi in cattle and swamp buffalo. J. Gem Microbial., 134: i77-181. Ho. Y.W., Bauchop, T., Abdullah, N. and Jalaludin. S., 1990. RwniIloutJws clcgaw gen et sp nav., a polycentric anaerobic rumen fungus from cattle. Mycotaxon.. 38: 397-405. Jalaludin, S., J&n, 2.. Vijchulam P. and Len& R A., 1984. Urilization of palm press libre by the swamp buffalo. Paper presented at the Second Research Coordination Meeting ‘Isolope-
aided Studies on Non-protein Nitrogen and Agroindustrial By-products Utilization by Ruminants’. 27 Fetruarv-2 March 1984. Vienna (unoubl.1. Orpin, C.G., 1977% Invasionof plant tissue in the’romen by the flagellate Neocalli~~msn.~~ot~to/is. J. Gen. Microbial.. 98: 423-430. Orpin. C.G., 1977b. The rumen flagellaie Pworrwnos comnuotis. Its life-history and invasion of plant material in the rumen. J. Gen. Microbial., 99: 107-l 17. Orpin. C.G.. 1981. Fungi in ruminant degradation. In: Agricultural Science Seminar: Degradation ofPlam Cell-wall Material. Agricultural Rescarch Council. London, pp. 37-46. Orskov. E.R., de Hovell. F.D. and Mould. F.. 1980. The use of the nylon bag technique for the evaluation offeedstuffs. Trap. Anim. Prod., 5: 195-213. Phllhps, M-W., 1988. Unusual rumcn fungi isolated from oorthern Australian cattle and waler buffalo. In: J.V. Nolan, R.A. Leng and D.I. Demeyer (Editors), The Role of Protozoa and Fungi in Ruminant Digestion. Penembul Books, Armidale, pp. 247-249. Vijchulata, P.. Mahyuddin, M. and Sivarajasingam. S.. 1985. Effect of ammonium hydroxide treatment on nutritive value ofpalm press fibre in buffaloes and caltle. Malays. Appl. Biol.. 14: 13-17. Wuliji, T. and McManus, W.R.. 1988. Colonization of alkali-treated fibrous roughages by anaerobic rumen fungi. Asian-Au% J. Anim. Sci., I: 65-7 I.
ABSTRACT Ho, Y.W., Abdullah, N. and Jalaludin, S., 1991. Fungal colonizstionofricestraw and palm pressfibre in the rumen of cattle and buffalo. Awn. FeedScr. Technol.. 34: 3 l-321.
I
Fungal colonization and development in rice straw and palm pressfibre (mesacarp ofoil palm fruit after the extraction ofoil) were very similar in both cattle and buffalo. In both animal species,atlachmeat to rice straw by rumen fungal zoosporeswas rapid, within 15 min of umen incubation. At 6 h of mmen incubation, thin- and thick-walled tussles were colonized by fun@ hyphae and by 24 h fungal calonization was exIensive. Fungi were predominamlv thosewith spherical and ovoid smmmgia. Fusiform sporangia were in fcwcr numb&. Fungal colo&ation was also extensive at 48 and 72 h. The colonization and development ofthe fungi on the palm presstibre, both untreated and treated wilh ammonium hydroxide and ether. was slow.
INTRODUCTION
Studies on the anaerobic rumen fungi in the past 15 years have indicated that they are important colonizers of plant tibres (Orpin, 1977a, b, 1981; Bauchop, 1979a, b, 1981, 1989; Akin et al., 1983; Akin and Rigsby, 1987). They show a preference for the thick-walled lignitied cells, such as the sclerenchyma cells and vascular elements (Akin et al., 1983; Akin and Rigsby, 1987; Ho et al., 1988a) and have been reported to contributeconsiderably to the overall digestion of various forages and wheat straw (Akin et al., 1983; Gordon and Ashes, 1984; Akin and Rigsby, 1987). Low-quality roughages, such as fibrous crop residues, have in recent years been studied as a potential feed resource for ruminant animals in the tropics. However, knowledge on the digestion of these fibrous crop residues by rumen microbes, particularly the fungi, is lacking. The present study was carried out to examine the invasion and colonization by rumen fungi on two tibrous crop
0377-8401/91
I$0350
0 1991 Elsevier Science Publishers B.V. All rights reserved.
Y.W. HO ET AL.
3i?
residues, viz. rice straw and palm press tibre (mesocarp tibres of oil palm fruits after the extraction of oil), which are being studied as potential feed materials for cattle and buffaloes. MATERIALS
AND METHOCS
Two Kedah-Kelantan cattle (80s in&us) and two swamp buffaloes (Bubalus bubalis), approximately 18 months old, and all males, each with a permanent fistula in the rumen, were maintained in separate cages indoors. Each animal was fed once daily (O&30 h) with guinea grass (Panicum maximum) ad libitum and each had free access to mineral blocks (cobalt mineralized salt blocks) and water. Fibrous crop residues (rice strawand palm pressfibre) Rice straw and palm press fibre (PPF) were dried and ground through a 4 mm screen in a hammer mill. The rice straw used was untreated, but PPF, both untreated and treated with ether (for fat extraction) and ammonium hydroxide (NH&H) (for softening of fibres), was used. PPF was subjected to fat extraction (2-3 g per thimble) by refluxing with petroleum eiher (60-80°C) in a Soxhlet condenser reflux for 8 h and dried at 65°C for 48 h before use. The PPF contained 4% ether extract. For NH,OH treatment, PPF was incubated for 8 days with 3% NH,OH (v/v) at ambient temperature (Vijchulata et al., 1985). The above treatments of PPF were carried out to determine whether the removal of fat and softening of the fibres would improve colonization of the fibres by fungi. Chemical analyses of straw and PPF Chemical analyses for dry matter (DM), lignin and nitrogen were conducted according to standard procedures (Association of Official Analytical Chemists, 1980). Neutral detergent fibre (NDF) was determined following the method described by Goering and Van Soest ( 1970). Table 1 shows the chemical composition of straw and PPF, while Table 2 shows the in situ digestibility of straw, untreated PPF, ether-extracted PPF and ammonia-treated PPF determined by the nylon bag technique (Orskov et al., 1980). Incubation ofrice straw and PPF in the rumen About 1 g of material was placed separately in nylon bags (90x60 mm, mesh size SOpm). The bags were tied in the same manner as described by Ho
Chemical composition of straw and PPF Comoosition 1%)
SIIZW
PPF’
DM Crude protem NDF Lignin Ether extract
86.0 4.3 73.0 19.0 1.3
70.6 6.2 77.6 21.3 4.0
‘From previous studies on amm.xGa treatment of PPF. the chemical composition of the tihre listed above was not significantly changedby the treatment. TABLE 2 Percent DM loss at 48 h of rumen incubation of straw, and untreated and treated PPF Feed
Cattle
Buffalo
straw Untreated PPF Ether-extracted PPF Ammonia-treated PPF
49.3k5.6’ 20.7fO.7” 17.3kO.8” 35.s*o.7”
53.2i 1.4b 2i.6f0.2’ 19.6kO.6” 39.9+0.5b’
Means with different superscriptsin the same row differ significantly (PcO.05 ). ‘Jalaludin et at. (1984 ). et al. ( 19%) and were placed in the ventral section of the tumen. The bags with rice straw were incubated for I5 and 30 min, 1, 3,6, 24,48 and 72 h, and those with PPF for 6,24,48 and 72 h. After retrieval, the bags were washed with water and the samples fixed in appropriate solutions.
Preparation of samples for scanning electron and light microscopy Samples for scanning electron microscopy (SEM) and light microscopy observation were. prepared using the method described by Ho et al. (1988b). For every incubation period of each material in each animal, 15 fragments were studied with SEM and 20 fragments were observed with light microscopy. RESULTS AND DISCUSSION
Invasion and colonization of rice straw and PPF, untreated and treated with NH,OH and ether, by rumen fungi appeared similar in both cattle and buffalo. Table 3 shows the prevalence of fungal sporangia on each material at various incubation times En both species of animals. Rice straw fragments were observed to have more sporangia than PPF at the various incubation
Y.W.HO ET AL.
314 TABLE 3 Prevalenceof funpa! sporangia on rice straw and PPF Animal/malerials
lncubalion lime I5min
30min
Ih
3h
6h
24h
48 1
72h
Cattle
straw
+
t
+
+
++
+++
_
_
-
-
+tt
PPF PPF-E PPF-A
+ + +
+ + +
ct ++ ++
++ ++ ++ t+
+
+
+ -
+ _ _ -
++ + + +
+++ + + +
++t ++ ++ +t
++ tc +t t+
BUffal straw PPF PPF-E PPF-A
_
f, few sporangia; ++. moderate number of sporangia; +++, determmed. PPF-E, ether-extracted palm presslibre. PPF-A, ammonia-treated palm pressfiba.
abundant sporangia; -,
not
periods. The occurrence of sporangia on the treated and untreated PPF appeared to be very similar. Fungal zoospores were found to attach on rice straw fragments 15 min after rumen incubation and by 30 min many had germinated. Similar rapid attachment of zoospores to plant fragments in the rumen has been reported by Bauchop (1981) and Hc et al. (1988a). The zoospores attached predominantly on stomata, cracks, fissures and cm suifaces of the rice straw frag ments. Similar sites for attachment have been observed by other workers (Orpin, 1977b; Bauchop, 1979b; Akin et al., 1983; Ho et al., 1988a). By 1-3 h, some fungi had developed an extensive hyphal system in the thinwalled parenchyma cells of the rice straw. Some of the hyphae produced lobed or disc-shaped ‘appressoria’ with penetration pegs penetrating cell walls. Fungi with ‘appressoria’ colonizing guinea grass in the rumen of cattle and buffalo have been reported earlier by Ho et al. (1988a). After 6 h in the rumen, the thick-walled lignified tissues, such as sclerenchyma and vascular elements, were colonized by the fungi. Degradation of the cell wall occurred in the thinwalled tissues which were extensively colonized by fungi. Some degradation was also observed in the thick-walled tissues with profuse fungal colonization. Sporangia of the fungi were small, 14-18x 14-20 pm, mostly spherical or ovoid. At 24 h, fungal colonization of the straw fragments was very profuse (Fig. 1). Dense networks of hyphae were observed in the sclerenchyma and vascular tissues. Vascular cylinders were easily detached because of degraded tis-
315
Fig. I. (A) Fungal colonization of rice straw 24 h after incubation in the rumen of caltk. Bar. 30 pm. (B) An uncolonized rice straw fragment showir.g surface struci~~~; PH, prickle hair; PP, papillae; SC, silica crystals. Bar, 50 /urn. sues surrounding
them. Most of the fungal sporangia were much larger (40-150 pm diameter) than those observed at 6 h, and they were mostly spherical and ovoid. Fusiform and ellipsoidal sporangia were also present.
but in fewer numbers. Among the fungi which produced spherical or ovoid sporangia, some were polycentric species as several sporangia developed from a single hypha. Polycentric anaerobic rumen fungi have recently been isolated from cattle and buffalo fed a high-roughage diet in various parts of the world (Akin and Rigsby, 1987; Phillips, 1988; Barr et al., 1989; Ho et al., 1990). Fungi with fusiform sporangia were also recently reported to be polycentric species (Ho et al., 1990). By 48 h, much of the softer thin-walled tissues of the rice straw fragments were degraded, and the remaining sclerenchyma and vascular tissues were extensively colonized by fungi. Fungi with spherical or ovoid sporangia still predominated. However, large spherical and ovoid sporangia, like those present at 24 h, were much less numerous. Instead, a large number of smaller ovoid structures (8- 15 pm diameter) which could be germinated zoospores or young sporangia were present both outside and within the cells (Fig. 2). These young sporangia/germinated zoospores did not produce extensive networks of hyphae. Most of the hyphae produced were thick and short. Some of the larger sporangia appeared shrunken or collapsed with part of the sporangial walls disintegrater’ or with openings or pores in the walls (Fig. 3). These collapsed sporangia were similar to those observed in guinea grass by Ho et al. ( 1988a) in the rumen of cattle and swamp buffalo, and they are probably the remains of sporangial wall after the release of zoospores. Warty spores, which appeared similar to those reported by Ho et al. (1988a), were also
Fig. 2. Rice straw fragment 48 h after incubatiat in the rumqof buffalo wall ard small ovoid sporangia in the exposed cell lumina. Bar, 10 #tn.
showing
degraded cell
317
Fig. 3. Remains ofsporangia, after the liberation of zoospores, on a rice stmw fragment 48 h afterrumen incubation in cattle. Bar, 20~m.
observed in a few fragments. These warty spores, as suggested by Ho et al. ( 1988a), may be some kind of resting spores. Fungi with fusiform and ellipsoidal sporangia were more abundant at 48 h than at 24 h, but their numbers were still fewer than those with spherical or ovoid sporangia. These fungi were attached mainly to the vascular cylinders and their hyphae, often with constrictions, were seen colonizing the sclerenchyma and vascular tissues. Although the degradation of tissues was quite extensive at 72 h after rumen incubation, extensive fungal growth was not observed. The number of sporangia was very much less than that at 24 and 48 h. Sections of unincubated PPF showed that each strand of the PPF is made up of thick-walled, highly lignified sclerenchyma cells enclosing vascular tissues in the centre. Longitudinal files of silica cells with clusters of silica lie adjacent to the outermost layer of sclerenchyma cells. Where the silica cells are numerous, they form almost complete sheaths around the PPF strands. Treatment of PPF with ether or NH,OH did not seem to dislodge many of the silica clusters. Invasion and colonization by the rumen fungi on PPF untreated and treated with ether and with NH,OH appeared similar. The untreated PPF was colonized in the same manner as that treated with NH,OH or ether. Grenet and Barry ( 1988) also found that there was no difference in the colonization of rumen fungi on wheat straw untreated or treated with ammonia. Wuliji and McManus ( 1988) reported that pretreatment of sugar cane bagasse, oat straw and wheat straw with ammonia did not increase fungal colonization signifi-
318
Y.W.HO ET AL.
cantly, but for so;aum hay, oat hull and lucerne stem, fungal colonization was improved significantly by the pretreatment. At 6 h ofrumen incubation, fungal colonization on the PPF fragments was very limited. Germinated zoospores and small sporangia were observed on cut ends of fragments, damaged surfaces and cavities left by dislodged silica clusters (Fig. 4). Sporangia were mainly spherical and ovoid. Cylindrical and tiliform sporangia were in lower numbers. Bv 24 h, some fungal hyphae were observed in the lumina of the tibres, particularly near the cut ends. Some of the hyphae produced lobed or disclike ‘appressoria’. Sporangia produced by the fungi were variable in size and they were mostly spherical, ovoid, cylindrical and fusiform. The fusiform sporangia were large (45-130 longx 1S-55 pm wide), usua!ly with long sporangiophores (up to 140 pm long) raising the sporangia above the fragments (Fig. 5). At 48 and 72 h of rumen incubation, fungal hyphae were abundant in the lumina of the tibres. In areas with very profuse colonization by the fungi, some degradation was obsrrved in the cell wall of the fibres. The predominant fungi observed on the surface of the libres were those which produced fusiform sporangia. Small spherical or oval sporangia were observed mainly in cracks and crevices of the tibres (Fig. 6). The results of the present work showed that fungal colonization and devel-
Fig. 4. Germinated zoospore (2) in a cavity (C) left by dislodged silica clusten of a PPF, 6 h after ~umcn incubntion in cattle. S. !.ilica clusters. Bar, 5 pt.
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Fig. 5. A PPF showing a large fusiform sporangium with a long sporangiophore raising the sgorangium above the fibre, 24 h after rumen incubation in buffalo. Note the limited degradation of tissues. Bar, 20 pm.
Fig. 6. Small spherical or oval sporangia in cracks and crevices of the PPF, 48 h after rumen incubation in cattle. Note the top portion of the fibre which shows some silica cty~Ials and empty cavities left by dislodged crystals. Bar, 20~m.
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Y.W.HOETAL
opment seemed to be more abundant and rapid in the rice straw than in PPF (both untreated and treated with NH,OH and ether). Fungal colonization and development in both untreated and treated PPF was slow. Although there were significant differences between untreated PPF and PPF treated with NH,OH in percentage DM loss (Table 2), differences in the colonization and development of fungi in these untreated and treated tibres, as observed by SEM and light microscopy in this study, were not apparent. ACKNOWLEDGEMENTS
We thank Professor A. Nawawi, Department of Botany, University of Malaya, for his helpful suggestions, and Kasmawati Mahmood and Suleka Madhavan for their technical assistance and co-operation.
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