Transcript Ruminal anaerobic fungi: The potential plant

Ruminal anaerobic fungi:
The potential plant-fiber
degraders in the rumen
__________________________
Ravinder Nagpal
Dairy Microbiology Division,
National Dairy Research Institute,
Karnal 132001 (Haryana) India.
Introduction

Importance of ruminants to mankind has led to a great
deal of research into the digestion of plant fibers in
order to improve the production efficiency.

Majority of livestock subsist on poor quality fibrous
crop residues and agro-industrial wastes

Attempts are being made to enhance the digestibility of
poor quality feeds by various feed additives
contd…

Ruminal anaerobic fungi, an emerging group of
animal probiotics, account for upto 8% of the
microbial biomass in rumen and actively
colonize plant cell-walls

Gain access to plant materials that is
unavailable to other rumen microorganisms
contd…

Rhizoids of vegetative thalli penetrate plant tissues better
than bacteria and protozoa

Help in access of other rumen microbes to the secondary
cell wall of feed particles

More rapid and complete degradation of forage entering
the rumen through proficient fibre degrading enzymes

Development of direct-fed microbials (DFM) for
improved rumen performance appears to be a prerequisite for the sustainable animal production.
Rumen anaerobic fungi

Observed in the rumen as early as 1910, but were
believed to be flagellate protozoa
(Liebetanz et al, 1910; Braune et al, 1913)

Confirmed to be a true fungus by the presence of chitin
in its cell wall and designated as Neocallimastix frontalis
(Orpin, 1975)

Identified anaerobic fungi in sheep rumen, each of which
had a motile stage (the zoospore) and a non-motile
zoosporangium
(Orpin, 1975)

Found in the rumen and other parts of the gastrointestinal tract of herbivorous animals
(Williams et al, 1987; Rezaeian et al, 2004)

Play an active and positive role in fibre degradation as
evidenced by the presence of different hydrolytic
enzymes
(Williams et al, 1987; Samanta et al, 2001; Paul et al, 2003)

There is a significant reduction in in-vitro gas production
and degradation of fibrous feeds, indicating a positive
role played by fungi in fibre degradation
(Kamra et al, 2004; Lee et al, 2004; Dey et al, 2004)

The fibre-based diets stimulate the fungal growth in
the rumen of buffalo in comparison to diets rich in
easily fermentable carbohydrates
(Kamra et al, 2003)

These fungi prefer to get attached to the most
lignified tissues of plant feed material by producing
variety of enzymes
(Akin et al, 1987)
Classification

Based on ultrastructural characteristics of the zoospores,
anaerobic fungi were assigned to the order of
Spizellomycetales and in the family, Neocallimasticaceae
(Barr et al, 1988)

Suggested the subdivision of this family into three genera
containing monocentric species, Neocallimastix, Piromyces
(previously Piromonas) and Caecomyces (previously
Sphaeromonas)
(Gold et al, 1988)

Three polycentric genera have been described, Orpinomyces
(Barr et al, 1989), Anaeromyces (Breton et al, 1990) and Cyllamyces
(Ozkose et al, 2001).
Division:
Subdivision:
Class:
Order:
Family:
Genera:
Eumycota
Mastigomycotina
Chytridiomycetes
Spizellomycetales
Neocallimasticaceae
Monocentric:
Caecomyces: zoospores with one or two flagella; thallus
with a globular rhizoid
Neocallimastix: zoospore with four to twenty flagella;
thallus with filamentous branching rhizoids
Piromyces: zoospore with one to four flagella and
thallus with filamentous branching rhizoids
Polycentric:
Orpinomyces: multiflagellate zoospore
Anaeromyces: zoospore with one flagellum
Cyllamyces: zoospore with one to two flagella with
thalloid branched sporangiophore
Genus
Species
Source(s)
Reference(s)
Caecomyces
C. communis,
C. equi
Sheep
Horse
Gold et al, 1988
Gold et al, 1988
Piromyces
P. Communis
Sheep
Cow
Horse
Elephant
Ass
Deer
Goat
Horse
Donkey
Gold et al, 1988
Julliand et al, 1998
Li et al, 1990
Li et al, 1990
Breton et al, 1991
Ho et al, 1993
Ho et al, 1993
Gaillard-Martinie et al, 1995
Julliand et al, 1998
P. mae
P. dumbonica
P. rhizinflata
P. Minutus
P. Spiralis
P. citronii
Neocallimastix
N. frontalis
N. patriciarum
N. hurleyensis
N. variabilis
Sheep
Sheep
Sheep
Cow
Heath et al, 1983
Orpin and Munn, 1986
Webb and Theodorou, 1991
Ho et al, 1993
Anaeromyces
A. elegans
A. mucronatus
Cow
Sheep
Ho et al, 1993
Breton et al, 1990
Orpinomyces
O. joyonii
O. intercalaris
Sheep
Cow
Breton et al, 1989
Ho et al, 1994
Cyllamyces
C. aberensis
Cow
Ozkose et al, 2001
LIFE CYCLE

Life cycle lasts about 23-32 hours
(Joblin 1981; Bauchop 1983; Lowe et al., 1987)

The life cycle of monocentric fungi consists of an
alteration between a motile, zoosporic stage and a
vegetative, zoosporangial stage

Flagellate zoospores are released from a sporangium and
encyst by shedding their flagella

The cyst germinates to produce a germ tube, which later
develops into rhizoids
(Orpin et al, 1977)
Teunissen and Op den Camp, 1993; Harhangi, 2002
contd…

The development of zoospores from young sporangia may
occur within 8 hours after encystment under appropriate
conditions
(Orpin et al, 1977)

Polycentric fungi have indeterminate life cycles and are
not dependent upon the formation of zoospores for their
continued survival
(Ho & Bauchop, 1991)

Zoospores are produced infrequently or zoosporogenesis
is even absent
(Phillip et al, 1989)
Distribution

First isolation in the UK from the rumen of sheep
(Orpin, 1975)

Have been found on almost all the continents and in all
of the geographic regions, where there have been sought

Ubiquitous among ruminants such as cattle, buffalo,
goat
(Singhal et al, 2000; Dey et al, 2004; Thareja et al, 2006)
contd…

Red deer and impala
(Bauchop et al, 1979; Singhal et al, 2000)

Grey kangaroo, wallaroo and swamp wallaby
(Breton et al, 1989)

Fecal samples of hindgut fermenters such as ass, horse,
elephant and zebra
(Breton et al, 1990; Li et al, 1990)

Isolated from fecal and rumen samples of wild Neelgai
(Paul et al, 2004; Tripathi et al, 2007)
Isolation

Overlayering with partially molten agar with filtered
rumen fluid
(Orpin, 1975)

Plate culture technique for anaerobic fungi from rumen
digesta of sheep and cattle
(Lowe et al, 1985)

Roll-bottle method involving inoculating a dilution series
of molten agar medium with filtered rumen fluid
(Joblin, 1981)
Penicillin, Streptomycin, Neomycin and Chloramphenicol are added to the
isolation media to suppress the bacterial growth
Identification
Genus identification:
• Number of flagella per zoospore
• Rhizomycelium
• Shape of sporangium
(Breton et al, 1990; Asao et al, 1993)
contd…

Species are delimited on the basis of zoospore
ultrastructure
(Ho & Barr, 1995)

18 species in six genera have been classified in the
literature

Species identification by PCR-amplification and
sequencing of ITS1 and ITS2
(Brookman et al, 2000; Fliegerova et al, 2004)
Enumeration

Counts of individual zoospores and zoosporangia have
been used to estimate fungal populations in vitro and in
vivo
(Joblin, 1981; Ushida et al, 1989)

Used colony-forming units per gram dry weight of feces as
the basis for quantifying species of Piromyces.
(Breton et al, 1991)

Procedure based on the technique of most probable
numbers, was developed to enumerate rumen fungi as
thallus-forming units
(Theodorou et al, 1990)
Role of anaerobic fungi in fibre digestion

Role of rumen fungi in the degradation of plant fibre has
been examined extensively
(Lee et al, 2000; 2004; Samanta et al, 2001; Dey et al, 2004, Paul et al, 2004;
Thareja et al, 2006; Tripathy et al, 2007)

These fungi are better at penetrating plant tissue than
are bacteria and protozoa
(Orpin and Joblin, 1988)

Such penetration leads to faster and more complete
degradation of forage that enters the rumen.
(Bauchop and Mountfort, 1981)
contd…

Degradation of lignin-containing walls of plant cells is an
important characteristic of rumen fungi
(Mountfort et al, 1982; Akin and Benner, 1988)

Rumen fungi dissolve small amounts of phenolic
compounds from plant cell walls
(Orpin, 1983; Gordon et al, 1985)

Zoospores of many species appear to colonize the lignincontaining tissues preferentially and to establish colonies
localized on sclerenchyma and xylem cells.
(Akin et al, 1986)
contd…

Anaerobic fungi penetrate the cuticle, a barrier that
other microorganisms cannot cross.

Rumen fungi attack recalcitrant plant cell walls by
weakening the textural strength of the residue
(Akin et al, 1989, 1990)

The greater ability of rumen fungi to weaken forage
fibre may be important in enhancing forage utilization
by the host animal
(Borneman and Akin, 1990)
contd…

Increased digestibility of straw with use of different
anaerobic fungi viz., Orpinomyces, Piromyces and
Anaeromyces was observed
(Manikumar et al, 2002; Sehgal et al, 2002; Tripathy et al, 2007)

7-12% increase in voluntary intake of straw based diet
was reported when the sheeps were dosed with cultures
of monocentric fungi
(Gordon and Phillips, 1998)

Fungal culture increased Cellulose degradation by 26%
under in vitro environment.
(Lee et al, 2004)

Fungi degrades plant cell wall:
 Xylem and mestome bundle sheath in leaves
 Schlerenchyma ring in stem
 Cuticular barrier of leaves
(Bauchop et al., 1989)
Hydrolytic Enzymes

While rumen protozoa and bacteria have been shown to
play a role in plant fibre degradation
(Williams, 1988; Akin and Benner, 1988)

Rumen fungi display a somewhat greater potential for
the degradation of more heavily lignified plant tissues
(Akin et al, 1988)

To degrade and utilize plant cell walls, anaerobic fungi
produce a wide range of hydrolytic enzymes including:
contd…

Cellulases
(Barichievich and Calza, 1990; Yanke et al, 1993; Paul et al, 2004)

Hemicellulases
(Lowe et al, 1987; Mountfort and Asher, 1989)

Proteases
(Wallace and Joblin, 1985; Michel et al, 1993)

Amylases, Amyloglycosidases
(Mountfort and Asher, 1988; Paul et al, 2004)
contd…

Feruloyl and p-coumaryl esterases
(Borneman et al, 1990; 1991; 1992; Paul et al, 2004)

Various disaccharidases
(Hebraud and Fevre, 1988; Chen et al, 1994)

pectinases
(Gordon and Phillips, 1992)

Exonucleases or avicelases
(Cabe, 1998)
Interaction with other rumen microorganisms

Anaerobic fungi form quite stable cocultures with rumen
methanogenic bacteria as a result of their high
production of hydrogen
(Fonty and Joblin, 1991; Orpin and Joblin, 1997)

These cocultures produce an increased amount of fungal
biomass and exhibit an increase in both the rate and
extent of cellulose degradation
(Bernalier et al, 1989; 1991; Joblin et al, 1989)

Cellulolytic activity appeared to be inhibited, when
combined in coculture with the cellulolytic ruminococci
(Bernalier et al, 1992; Roger et al, 1993)
contd…

Growth of the rumen fungi was found to be markedly
inhibited in cocultures with rumen bacteria
(Dehority and Tirabasso, 2000)

Coincubation of protozoa with fungi has shown that the
protozoa are able to both ingest and digest fungi
(Orpin and Joblin, 1997)

Chitinase activity in samples of mixed rumen protozoa
account for their predatory activity on the rumen fungi
(Joblin, 1990; Williams et al, 1994; Morgavi et al, (1994)
Potential benefits of ruminal anaerobic fungi
for improved animal nutrition and productivity

Improved fibre digestion and nutrient
utilization

More feed intake and feed efficiency

Increased body weight

Improved milk production
Prospective applications of
ruminal anaerobic fungi
Could be exploited as:

Direct-fed microbials

Animal feed additives

Novel silage inoculants

For large scale production of enzymes (e.g.
cellulase)