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Philippine Journal of Science
145 (2): 153-164, June 2016
ISSN 0031 - 7683
Date Received: ?? Feb 20??
Key words: Barnyard grass, Dematiaceae, Echinochloa glabrescens, endophytic fungi, Nueva
Ecija, rice, Oryza sativa
Identities, Characteristics, and Assemblages of
Dematiaceous-Endophytic Fungi Isolated
from Tissues of Barnyard Grass Weed
1Crop Protection Division, Philippine Rice Research Institute
Central Experiment Station, Maligaya, Science City of Muñoz, 3119 Nueva Ecija
2Crop Protection Cluster, College of Agriculture,
University of the Philippines Los Baños, College, 4301 Laguna
*Corresponding author: dindoking08@gmail.com
dkm.donayre@philrice.gov.ph
Dindo King M. Donayre1 and Teresita U. Dalisay2
Barnyard grass weed (Echinochloa glabrescens) in ricefields of Nueva Ecija, Philippines had
been reported to harbor different isolates of endophytic fungi. Despite the discovery that its
tissues are hosts to diverse endophytic fungi, the identities, characteristics, and assemblages,
particularly of the dematiaceous-endophytic fungi, are still unknown. This paper, thus, aimed
to identify, and describe the characteristics and assemblages of the different dematiaceous-
endophytic fungi that were isolated from tissues of barnyard grass weed of Nueva Ecija,
Philippines. Microscopic examination revealed that there were eight common genera of
dematiaceous-endophytic fungi residing in tissues of barnyard grass weed namely, Alternaria,
Arthrinium, Bipolaris, Curvularia, Nigrospora, and Stemphyllium along with the two unidentified
coded genera. Further examination of the taxonomic characteristics and assemblages showed
that there were two species in the genus of Alternaria (A. alternate and A. tenuissima) while four
for Bipolaris (B. australienses, Bipolaris sp. EF-ds102, B. avenacea, and Bipolaris rostrata), four
for Curvularia (C. lunata, C. prasidii, C. pallescens, and Curvularia sp. EF-ds427), and two for
Nigrospora (N. oryzae and Nigrospora sp. EF-ds180).
INTRODUCTION
Endophytic fungi are organisms that are capable of
living inside plant tissues without causing harm to their
hosts (Caroll 1988; Petrini 1991; Stone et al. 2004).
Although they may form other interrelationships such
as commensalism and latent/quiescent parasitism,
their interrelationships with their hosts are generally
mutualistic, that is, the host gains while the endophytic
fungi are also gaining (Clay 1991; Backman and Sikora
2008). Endophytic fungi have been reported to have
many uses. Hence, explorations about their ecological
role, beneficial uses for human and animal health; and
growth promotions, drought tolerance, and protection
of crops against pests are widely increasing around
the world (Arnold et al. 2003; Evans 2003; Tian et al.
2004; Kuldau & Bacon 2008; Ting et al. 2008; Ahmad
et al. 2010, Hipol 2012, Bungihan 2013 et al., Tan et al.
2015; Eskandarighadikolaii et al. 2015). Barnyard grass
(Echinochloa glabrescens Munro ex Hook. f.), on the
other hand, is one of the major weeds of rice (Michael
1978; Moody 1989; Pancho & Obien 1995; Caton et al.
2010). It is an erect, tufted, highly competitive annual
grass that grows up to 1m high under lowland ricefield
153
conditions. At 5 to 40% level of infestation in the field,
barnyard grass weed can significantly reduce the yield of
rice from 6 to 73% by way of competition (Rao & Moody
1992). Aside from being a competitor, barnyard grass is
also an alternate host to different pests of rice like the green
leafhopper [Nephotettix virescens (Distant)], tungro and
ragged stunt viruses (Rice tungro bacilliform virus, Rice
tungro spherical virus, and Rice ragged stunt virus), rice
black bug [Scotinophara latiuscula (Breddin)], and golden
apple snail (Pomacea canaliculata Lam.) (Salamat et al.
1987; Khan et al. 1991; Pancho & Obien 1995; Joshi et al.
2006; Litsinger 2007). Despite the negative impact to rice
production, however, barnyard grass had been reported
to harbor beneficial organisms like the endophytic fungi.
In exploring diversity and tissue specificity, Donayre et
al. (2014) recovered numerous endophytic fungal isolates
from tissue segments of barnyard grass weed that were
collected around ricefields of Nueva Ecija, Philippines.
Among the isolates, the hyphomycetes, particularly
the dematiaceous (dark-colored) ones, were the most
frequently isolated endophytic fungal group. Although
the authors discovered that tissues of barnyard grass had
very diverse endophytic fungal community, the identities,
characteristics, and assemblages of dematiaceous-
endophytic fungi have never been described in the report.
Hence, this paper primarily aimed mainly to identify
and describe the characteristics and assemblages of the
different dematiaceous-endophytic fungi isolated from
tissues of barnyard grass weed of Nueva Ecija, Philippines.
MATERIALS AND METHODS
Some of the methods described for weed collection and
isolation of endophytic fungi in this paper can also be
found in the report of Donayre et al. (2014). Healthy-
looking barnyard grass plants at their reproductive stages
were collected in lowland ricefields of 16 towns of Nueva
Ecija, Philippines. In each field, three individual barnyard
grass plants were randomly collected and brought into
the Plant Pathology Laboratory of the Crop Protection
Division, Philippine Rice Research Institute Central
Experiment Station, Maligaya, Science City of Muñoz,
Nueva Ecija. Coordinates of the fields where collection of
barnyard grass plants were conducted were also recorded
using a global positioning system tool (Garmin: Etrex
Summit). In the laboratory, seeds, leaves, leaf sheaths,
and roots of barnyard grass were sampled for isolation
following the methods used by Schulz et al. (1993) and
Stone et al. (2004). Portions of each tissue except the seeds
and roots were cut into 2 x 2 mm sections in a “Z” pattern.
For the roots, 2 mm segments were randomly cut from
each plant sample. Twenty pieces of seeds, on the other
hand, were randomly selected from each plant sample. All
tissue sections were surface-sterilized by immersing them
in 95% ethanol for 30 sec, 10% NaOCl for 5 min, and
95% ethanol for 30 sec. Tissue sections were then rinsed
three times in sterile distilled water and blotted dry in a
clean-sterilized tissue paper. Five representatives from
each sampled tissues were planted on previously cooled
malt extract agar (Pronadisa, 33 g/L H2O). The plates were
incubated in an inverted position for 5 to 10 days at room
temperature of 23-27 0C. Agar bits with mycelial growth
of each endophytic fungus was separately transferred into
1.5 ml capacity sterile micro-centrifuge tubes previously
filled with sterilized potato dextrose agar (Pronadisa, 39
g/L H2O). Slide mounts of each endophytic fungal isolate
were prepared through an agar-block technique under
laboratory conditions of 22 oC at alternating 12 hours light
and dark (Dhingra and Sinclair 1995). In each slide, 7 to
50 conidia and conidiophores were randomly selected for
capturing and measuring the images under a microscope
digital camera (Olympus DP72-BSW). Captured images
of endophytic fungal isolates were then identified and
characterized by comparing them with images that were
found in the works of Barron (1968); Rayner (1970); Ellis
(1971); Sun & Zhang (2008); Pitt & Hocking (2009);
Mew & Gonzales (2002); Watanabe (2010); and Santiago
et al. (2011).
RESULTS
Microscopic examination of each isolate using available
images revealed that there were eight common genera
of dematiaceous-endophytic fungi residing in the tissues
of barnyard grass weed of Nueva Ecija, Philippines.
The genera were identified as Alternaria, Arthrinium,
Bipolaris, Curvularia, Nigrospora, and Stemphyllium.
Two coded genera, on the other hand, were still unknown.
Further examination of the taxonomic characteristics and
assemblages also revealed that there were two species in
the genus of Alternaria (A. alternate and A. tenuissima)
while four for Bipolaris (B. australienses, Bipolaris sp.
EF-ds102, B. avenacea, and Bipolaris rostrata), four for
Curvularia (C. lunata, C. prasidii, C. pallescens, and
Curvularia sp. EF-ds427), and two for Nigrospora (N.
oryzae and Nigrospora sp. EF-ds180). The taxonomic
characteristics, assemblages, and other special features
of each species were the following:
Alternaria alternata (Fr.) Keissler
Descriptions and dimensions of assemblages:
conidiophores brown, simple, bearing catenate conidia
at the apex; conidia with beaks, brown, obclavate,
branched, catenate, mostly 2 to 6 in chain, composed
of 1-6 transverse walls and 1-4 longitudinal walls; beak
brown, long, slightly geniculate (Figure 1); conidiophores
Donayre and Dalisay: Identitites, Characteristics, and
Assemblages of Dematiaceous-Endophytic Fungi
Philippine Journal of Science
Vol. 145 No. 2, June 2016
154
198.23 - 239.16 x 8.85 - 12.56 µm (x=210.61 x 10.25 µm,
ñ=25), conidia 40.75 - 88.02 x 14.67 - 20.75 µm (x=62.64
x 17.72 µm, ñ=25), beak 0.75-56.21 x 0.08-5.75 µm
(x=24.24 x 17.72 µm, ñ=25). Cultural characteristics
in potato dextrose agar (PDA): colony growth 69 mm
(diameter) after 10 days of incubation; medium dense,
smooth, round with scalloped margin, umbonate, pale
greenish grey in color (obverse); dark brick at the center
with brick to rosy buff margin (reverse). Source of tissue:
seed. Source of barnyard grass: Brgy. Pinili, San Jose,
Nueva Ecija, Philippines; N 150 45' 22.3", E 1210 01' 10.1''.
References: Rayner (1970); Ellis (1971); Sun & Zhang
(2008); Pitt & Hocking (2009).
Alternaria tenuissima (Kunze) Wiltshire
Descriptions and dimensions of assemblages:
conidiophores dark brown, simple, bearing catenate
conidia at the apex; conidia with short beaks, dark
brown, obclavate, no branch, catenulate, mostly 1 to 8
in chain, fusiform composed of 3-4 transverse and
1-2 longitudinal walls; beak brown, short (Figure 1);
conidiophores 200.23 – 241.16 x 10.65 – 14.56 µm
(x=212.0 x 12.36 µm, ñ=25); conidia 42.75 – 90.02 x
16.67 – 22.75 µm (x=65.65 x 19.97 µm, ñ=25); beak
0.44 – 33.02 x 0.4 – 4.75 µm (x=11.6 x 2.28 µm, ñ=25).
Cultural characteristics in PDA: colony growth 61.5 mm
(diameter) after 10 days of incubation; medium dense,
smooth, round, raised, pale luteos to luteos in color
(obverse and reverse). Source of tissue: upper midrib,
lower leaf (near basal stem). Source of barnyard grass:
Brgy. San Pedro, Sta. Rosa, Nueva Ecija, Philippines; N
150 26' 03.7", E 1200 53' 54.6''. References: Rayner (1970);
Ellis (1971); Sun & Zhang (2008); Pitt & Hocking (2009).
Arthrinium pheospermum (Corda) M.B. Ellis
[Syn: Papularia sphaerosperma (Pers.)Höhn]
Descriptions and dimensions of assemblages:
conidiophores simple, smooth, erect, thin, hyaline;
conidia dark brown, one-celled, ellipsoidal, occasionally
subglobose and reniform, with narrow hyaline band
around the longest periphery (Figure 2); conidiophores 0.9
– 10.17 x 0.39 – 1.34 µm (x=3.6 x 0.74 µm, ñ=27), conidia
2.05 – 6.49 x 1.31 – 2.56 µm (x=2.66 x 1.89, ñ=50).
Cultural characteristics in PDA: colony growth 57.5 mm
(diameter) after 7 days of incubation; dense, lobate, round
with radiating margins, convex (obverse); entirely white,
49.5 mm in diameter after 7 days of incubation (reverse).
Source of tissue: middle leaf blade, upper leaf (near flag
leaf). Source of barnyard grass: Brgy. San Pedro, Sta.
Rosa, Nueva Ecija, Philippines; N 150 26' 03.7", E 1200
Figure 1. Alternaria alternata - assemblages (a-f), colony growth in PDA at 10 days after incubation (g-h), scale bars =
30μm; A. tenuissima - assemblages (i-k), colony growth in PDA at 10 days after incubation (l-m), scale bars
= 20μm (i-j) and 10μm (k).
Donayre and Dalisay: Identitites, Characteristics, and
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Philippine Journal of Science
Vol. 145 No. 2, June 2016
155
53' 54.6''. References: Barron (1968); Rayner (1970); Ellis
(1971); Mew & Gonzales (2002); Pitt & Hocking (2009);
Watanabe (2010).
Bipolaris australienses (M.B. Ellis) Tsuda. & Ueda
Descriptions and dimensions of assemblages:
conidiophores brown, simple, bearing conidia apically
and laterally; conidia brown, oval to ovoid, 4-celled, with
hilum at the base (Figure 3); conidiophores 69.31 - 239.93
x 7.99 - 80.32 µm (x=166.55 x 11.71 µm, ñ=50), conidia
16.33 - 52.37 x 16.16 - 22.03 µm (x=42.93 x 19.01 µm,
ñ=50). Cultural characteristics in PDA: colony growth 44
mm (diameter) after 3 days of incubation; medium dense,
round with scalloped margin, raised, olivaceous grey
with white, olivaceous black to orange margin (obverse
and reverse). Source of tissue: upper midrib, upper leaf
(near flag leaf). Source of barnyard grass: San Leonardo,
Nueva Ecija, Philippines; N 150 26' 03.7", E 1200 53' 54.6''.
References: Rayner (1970); Ellis (1971); Watanabe (2010).
Bipolaris sp. EF-ds102
Descriptions and dimensions of assemblages: conidiophores
brown, smooth, erect, simple, bearing 1 to 2 conidia only at
the apical portion; conidia brown, ellipsoidal, 4-celled, with
hilum at the base (Figure 3); conidiophores 23.28 - 258.62
x 7.65 - 9.78 µm (x=133.46 x 358.62 µm, ñ=50); conidia
46.70 - 66.62 x 17.21 - 20.06 µm (x=55.76-18.47 µm,
ñ=50). Cultural characteristics in PDA: colony growth 59
mm (diameter) after 6 days of incubation, medium dense,
irregular, raised, olivaceous to olivaceous grey (obverse);
Figure 2. Arthrinium pheospermum - assemblages (a-c), colony growth in PDA at 10 days after incubation (d-e); scale
bars = 5μm (a-c).
Donayre and Dalisay: Identitites, Characteristics, and
Assemblages of Dematiaceous-Endophytic Fungi
Philippine Journal of Science
Vol. 145 No. 2, June 2016
156
olivaceous black with orange margin (reverse) (Figure 9).
Source of tissue: bottom leaf blade, upper leaf (near flag
leaf). Source of barnyard grass: Brgy. Campus, Talavera,
Nueva Ecija, Philippines; N 150 36' 41.5", E 1200 55' 25.9''.
References: Rayner (1970); Ellis (1971).
Bipolaris avenacea (Curtes ex Cooke) Shoemaker
Descriptions and dimensions of assemblages: conidiophores
pale brown, rough, slightly curved, simple, bearing 6 to
12 conidia apically and laterally; conidia pale brown,
ellipsoidal to sub-cylindrical, consist of 4 to 6 cells, with
hilum at the base (Figure 3); conidiophores 23.68 - 232.50
x 7.28 - 11.90 µm (x=70.67 x 9.53 µm, ñ=31); conidia
43.33 - 79.06 x 13.16 - 21.17 µm (x=61.91 x 17.30 µm,
ñ=50). Cultural characteristics in PDA: colony growth 57
mm (diameter) after 6 days of incubation, dense, entirely
smooth, round, convex, lavender grey (obverse); olivaceous
black (reverse). Source of tissue: middle midrib, lower leaf
(near basal stem). Source of barnyard grass: Brgy. Campus,
Talavera,Nueva Ecija, Philippines; N 150 36' 41.5", E 1200
55' 25.9''. References: Rayner (1970); Ellis (1971).
Figure 3. Bipolaris australienses - assemblages (a-c), colony growth in PDA at 3 days after incubation of (l-m), scale
bars = 10μm; Bipolaris sp. EF-ds102 - assemblages (d-f), colony growth in PDA at 6 days after incubation
(n-o), scale bars = 20μm; B. avenacea - assemblages (g-h), colony growth in PDA at 6 days after incubation
(p-q), scale bars = 60μm (g) and 20μm (h); B. rostrata - assemblages (i-k), colony growth in PDA at 10 days
after incubation (r-s), scale bars = 30μm (i-j) and 50 μm (k).
Donayre and Dalisay: Identitites, Characteristics, and
Assemblages of Dematiaceous-Endophytic Fungi
Philippine Journal of Science
Vol. 145 No. 2, June 2016
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Bipolaris rostrata (Drechsler) Shoemaker
Descriptions and dimensions of assemblages:
conidiophores pale brown, smooth, branched, bearing 1
to 2 conidia apically and laterally; conidia pale brown,
fusoid to ellipsoidal, consist of 5 to 7 cells, with hilum
at the base (Figure 3); conidiophores 205.30 - 468.58 x
10.37 - 15.67 µm (x=300.0 x 12.99 µm, ñ=18); conidia
41.25 - 175.33 x 23.28 - 32.79 µm (x=107.79 x 27.31 µm,
ñ=18). Cultural characteristics in PDA: colony growth
40 mm (diameter) after 10 days of incubation; medium
dense, irregular, raised, pale olivaceous grey (obverse);
olivaceous black (reverse). Source of tissue: upper leaf
blade, upper leaf (near flag leaf). Source of barnyard
grass: Brgy. Pinili, San Jose, Nueva Ecija, Philippines;
N 150 45' 22.3", E 1210 01' 10.1''. References: Rayner
(1970); Ellis (1971).
Curvularia lunata (Walker) Boedijn
Descriptions and dimensions of assemblages:
conidiophores dark brown, curved, occasionally
geniculate, simple, bearing conidia apically and laterally
with conspicuous pores left after spore detachment;
conidia dark brown, curved, sub-ellipsoid, occasionally
oval to ovoid, 4-celled, darker brown in 2-central cells,
with distinct hilum basally (Figure 4); conidiophores 25.73
- 658.04 x 0.26 - 9.65 µm (x=304.45 x 2.66 µm, ñ=50);
conidia dark brown, curved, sub-ellipsoid, occasionally
oval to ovoid, 4-celled, darker brown in 2-central cells,
with distinct hilum basally. Cultural characteristics in
PDA: colony growth 80 mm (diameter) after 6 days of
incubation; smooth, irregular, flat, mouse grey (obverse);
mouse grey to olivaceous black (reverse). Source of tissue:
middle leaf blade, lower leaf (near basal stem). Source of
barnyard grass: Brgy. San Pedro, Sta. Rosa, Nueva Ecija,
Philippines; N 150 26' 03.7", E 1200 53' 54.6''. References:
Rayner (1970); Ellis (1971); Pitt & Hocking (2009);
Watanabe (2010); Santiago et al. (2011).
Curvularia prasidii R.L. & B.L. Mathur
Descriptions and dimensions of assemblages:
conidiophores brown, rugose, geniculate, simple, bearing
apical and lateral conidia distantly; conidia brown, curved,
ellipsoidal, 4-celled, darker brown in 2-central cells
(Figure 4); conidiophores 101.51 - 188.50 x 6.73 - 13.44
µm (x=148.19 x 9.93 µm, ñ=50); conidia 38.92 - 61.73 x
14.61 - 28.25 µm (x=49.37 x 20.97 µm, ñ=50). Cultural
characteristics in PDA: colony growth 71.5 mm (diameter)
after 6 days of incubation; thin, wavy, almost irregular,
flat, smoke grey in color (obverse and reverse). Source
of tissue: upper leaf sheath (near flag leaf). Source of
barnyard grass: San Leonardo, Nueva Ecija, Philippines;
N 150 22' 20.3", E 1200 50' 19.7''. References: Rayner
(1970); Ellis (1971).
Curvularia pallescens Boedijn
Descriptions and dimensions of assemblages:
conidiophores dark brown, rugose, geniculate, simple,
bearing apical and lateral conidia in whorls; conidia
dark brown, curved, ellipsoid, oblong to ovoid, 4-celled,
darker brown in 2-central cells (Figure 4); conidiophores
11.06 - 26.55 x 0.35 - 0.60 µm (x=23.58 x 0.48 µm,
ñ=17); conidia 4-celled, darker brown in 2-central cells,
13.21 - 20.21 x 6.02 - 9.91 µm (x=16.60 x 8.05 µm,
ñ=50). Cultural characteristics in PDA: colony growth
66.5 mm (diameter) after 6 days of incubation; thin,
wavy, flat, greyish sepia in color (obverse and reverse).
Source of tissue: lower leaf sheath (near basal stem).
Source of barnyard grass: Brgy. San Pedro, Sta. Rosa,
Nueva Ecija, Philippines; N 150 26' 03.7", E 1200 53'
54.6''. References: Rayner (1970); Ellis (1971); Pitt &
Hocking (2009); Santiago et al. (2011).
Curvularia sp. EF-ds427
Descriptions and dimensions of assemblages:
conidiophores brown, geniculate, simple, bearing
lateral conidia; conidia brown, curved, ellipsoid to
sub-cylindrical, 4-celled, cells evenly colored (Figure
4); conidiophores 134.53 - 299.87 x 6.02 - 10.33 µm
(x=190.14 x 8.26 µm, ñ=7); conidia 41.31 - 60.38 x
16.77 - 31.43 µm (x=51.10 x 22.79 µm, ñ=50). Cultural
characteristics in PDA: colony growth 33.5 mm (diameter)
after 10 days of incubation; thin, lobate, flat, mouse
to pale mouse grey (obverse); dark mouse grey with
concentric rings (reverse). Source of tissue: middle leaf
blade, lower leaf (near basal stem). Source of barnyard
grass: Sanggalang, Jaen, Nueva Ecija, Philippines; N 150
23' 06.9", E 1200 50' 29.3''. References: Rayner (1970);
Ellis (1971).
Nigrospora oryzae (Berk. & Broome) Petch
Descriptions and dimensions of assemblages:
conidiophores hyaline, short, simple, inflated below the
tip, bearing single conidia apically; conidia dark, sub-
globose to globose, smooth, 1-celled, borned apically,
solitary (Figure 5); conidiophores 11.14-26.28 x 11.42-
18.64 µm (x=20.51 x 14.18 µm, ñ=6), conidia 22.27-26.62
x 24.02-38.17 µm (x=31.21 x 31.27 µm, ñ=20). Cultural
characteristics in PDA: colony growth 53.5 mm (diameter)
after 4 days of incubation; dense, irregular, convex, pale
olivaceous grey (obverse); olivaceous black at the center
to olivaceous grey. Source of tissue: middle leaf blade,
upper leaf (near flag leaf). Source of barnyard grass: Brgy.
Poblacion, Rizal, Nueva Ecija, Philippines; N 150 42'
03.8", E 1210 05' 27.5''. References: Rayner (1970); Mew
& Gonzales (2002); Pitt & Hocking (2009); Watanabe
(2010); Santiago et al. (2011).
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Philippine Journal of Science
Vol. 145 No. 2, June 2016
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Figure 4. Curvularia lunata - assemblages (a-b), colony growth in PDA at 6 days after incubation (k-l), scale bars =
10μm; C. prasidii - assemblages (c-e), colony growth in PDA at 6 days after incubation (m-n), scale bars =
25μm; C. pallescens - assemblages (f-h), colony growth in PDA at 6 days after incubation (o-p), scale bars
= 60μm (f) and 10μm (g-h); Curvularia sp. EF-ds427 - assemblages (i-j), colony growth in PDA at 10 days
after incubation (q-r), scale bars = 30μm.
Nigrospora sp. EF-ds180
Descriptions and dimensions of assemblages:
conidiophores hyaline, short, simple, tapering, inflated
below the tip, bearing solitary conidia; conidia dark,
sub-globose, smooth, 1-celled, borned apically, solitary
(Figure 5); conidiophores 0.73 – 1.70 x 0.60 – 1.06 µm
(x=1.18 x 0.86 µm, ñ=13), conidia 0.62 – 1.58 x 0.71 –
1.75 µm (x=1.19 x1.36 µm, ñ=18). Cultural characteristics
in PDA: colony growth 52 mm (diameter) after 10 days
of incubation; thin, wavy, round with radiating margin,
raised, white at the center with olivaceous grey margin
(obverse); olivaceous grey (reverse). Source of tissue:
middle leaf blade, upper leaf (near flag leaf). Source
of barnyard grass: Brgy. Imama, llanera, Nueva Ecija,
Philippines; N 150 39' 15.1", E 1210 01' 58.1''. Reference:
Rayner (1970).
Donayre and Dalisay: Identitites, Characteristics, and
Assemblages of Dematiaceous-Endophytic Fungi
Philippine Journal of Science
Vol. 145 No. 2, June 2016
159
Stemphylium botryosum Wallroth
Descriptions and dimensions of assemblages:
conidiophores brown erect, simple with nodal formation,
bearing single conidia. Conidia brown, solitary, ovoid to
cylindrical with rounded end, 2-celled when immature,
muriform when mature with 1-3 longitudinal septa and 1-5
transverse septa (Figure 6); conidiophores 15.64 – 432 x
2.28 – 9.46 µm (x=73.25 x 5.19 µm, ñ=50); conidia 2.10
– 39.98 x 5.84 – 90.0 µm (x=21.38 x 12.66 µm, ñ=50).
Cultural characteristics in PDA: colony growth 33.5 mm
(diameter) after 10 days of incubation; medium dense,
smooth, round, convex, white (top view); saffron to pale
luteous (back view). Source of tissue: upper midrib, older
lower leaf (near basal stem). Source of barnyard grass:
Brgy. Campus, Talavera, Nueva Ecija, Philippines; N 150
36' 41.5", E 1200 55' 25.9''. References: Watanabe (2010),
Pitt & Hocking (2009), Barron (1968), Rayner (1970).
EF-ds68-129
Descriptions and dimensions of assemblages: conidiophore
thin, pale brown, and smooth; conidia pale to dark brown,
pyriform, 1-2 celled, and catenate; chlamydospores ellipsoidal,
occasionally pyriform, and catenate (Figure 7); conidiophore
1.15 – 18.65 x 0.3 – 0.43 µm (x=6.69 x 0.38 µm, ñ=5); conidia
0.53 – 1.80 x 0.43 – 1.36 µm (x=1.0 x 0.67 µm, ñ=25);
chlamydospores 0.43 – 1.44 x 0.45 – 0.98 µm (x=1.03 x 0.69
µm; ñ=10). Cultural characteristics in PDA: colony growth 83
mm (diameter) after 3 days of incubation; dense, smooth, round
with radiating margin, white (obverse); white with olivaceous
buff at the center (reverse). Source of tissue: middle leaf blade,
upper leaf (near flag leaf). Source of barnyard grass: Brgy.
Burgos, Sto. Domingo, Nueva Ecija, Philippines; N 150 37'
04.3”, E 1200 52' 37.8''. Reference: Rayner (1970).
EF-ds392
Descriptions and dimensions of assemblages:
conidiophore pale brown, curved, and inflated at the
middle; conidia dark brown, allantoid, reniform, and 2-4
celled (Figure 7); conidiophore 0.41 – 1.66 x 0.16 – 0.73
(x=0.92 x 0.37 µm, ñ=7); conidia 0.48 – 2.16 x 0.52 – 1.29
µm (x=1.10 x 0.80 µm, ñ=20). Cultural characteristics in
PDA: colony growth 76.5 mm (diameter) after 20 days of
incubation; dense, irregular, raised, pale mouse to mouse
grey (obverse); umber (or amber?) to ochreous (reverse).
Source of tissue: roots. Source of barnyard grass: Brgy.
Carmen, Zaragosa, Nueva Ecija, Philippines; N 150 27'
13.4", E 1200 49' 49.3''. Reference: Rayner (1970).
DISCUSSION
Dematiaceous fungi are known for their typical dark
hyphae and conidia (Alexopoulus & Mims 1979). These
fungi that do not produce fruiting bodies are one of the
most encountered endophytes in many tissue parts of
different plants of the world. It has been suggested that
the occurrence and dominance of dematiaceous fungi in
many plants are due to their conidial structure. Unlike other
groups of fungi where spores are born inside specialized
structures like ascus, perithecium, and basidium, the spores
(also known as conidia) of Hyphomycetes, particularly the
members of the Dematiaceae, are born naked. Because of
this, their exposed-spores are highly vulnerable to actions
of dispersing agents like wind, rain splashes, or even
humans and animals. Numerous studies had already been
reported about the occurrence, dominance and presence of
dematiaceous fungi in particular tissues of plants. One of
Figure 5. Nigrospora oryzae - assemblages (a-b), colony growth in PDA at 4 days after incubation (c-d), scale bars = 40μm;
Nigrospora EF-ds180 - assemblages (e-g), colony growth in PDA at 10 days after incubation (h-i), scale bars = 2μm.
Donayre and Dalisay: Identitites, Characteristics, and
Assemblages of Dematiaceous-Endophytic Fungi
Philippine Journal of Science
Vol. 145 No. 2, June 2016
160
Figure 6. Stemphylium botryosum - assemblages (a-e), colony growth in PDA at 10 days after incubation
(f-g), scale bars = 10μm.
Figure 7. EF-ds68129 - assemblages (a-d), colony growth in PDA at 3 days after incubation (e-f), scale
bars = 2μm; EF-ds392 - assemblages (g-i), colony growth in PDA at 20 days after incubation
(j-k), scale bars = 2μm.
Donayre and Dalisay: Identitites, Characteristics, and
Assemblages of Dematiaceous-Endophytic Fungi
Philippine Journal of Science
Vol. 145 No. 2, June 2016
161
these was the work of Donayre et al. (2014) on the diversity
and tissue specificity of endophytic fungi in barnyard
grass weed (Echinochloa glabrescens Munro ex Hook.
f.) where the dematiaceous fungi that had been recovered
were characterized in this paper. Exploring the distribution
of endophytic fungi in lemon grass [Cymbopogon citratus
(DC) Stapf.], Deshmukh et al. (2010) also recovered
and identified dark-septated fungi namely, Alternaria
alternata (Fr.) Keisler (1-3.5% frequency of colonization),
Arthrinium phaeospermum (Corda) M.B. Ellis (0.5%),
Drechslera sp. (4.5-6.5%), Nigrospora sphaerica (Sacc.)
Mason. (0.5-1%), and N. oryzae (Berk. & Broome) Petch
(0.5%). Likewise, Sadoral (2010) explored the healthy
leaves of Jatropha curcas L. and recovered and identified
Bipolaris sp. In separate studies conducted by Lanceta
(2010) and Cristobal (2010), they also recovered genera
of dematiaceous fungi namely, Bipolaris, Curvularia,
Nigrospora, and Thielaviopsis from healthy leaf tissues
of irrigated-lowland and upland rice plants (Oryza sativa
L.). Similar findings were also obtained by Ramirez
and Dela Cruz (2011) on mangrove plants where they
recovered Nigrospora sp.; Geris dos Santos (2003) on
Melia azedarach L. (Nigrospora sp.); Larran et al. (2007)
on wheat [A. alternata, A. infectoria species-group,
Arthrinium sp., Bipolaris sorokiniana (Sacc.) Shoem., B.
spicifera (Bainier) Subramanian, Bipolaris sp., Curvularia
lunata (Wakker) Boedijni, Nigrospora sp., Stemphylium
botryosum Wallr.]; and Larran et al. (2001) on tomato [A.
alternata, Arthrinium sp., Bipolaris cynodontis (Marig.)
Shoem., N. sphaerica, and S. botryosum].
CONCLUSION
There were eight common genera of dematiaceous-
endophytic fungi residing in tissues of barnyard grass weed
of Nueva Ecija. Except for Stemphyllium and the unknown
genera, each genus of dematiaceous-endophytic fungus had
more morpho-species based on taxonomic characteristics,
assemblages, and other special features. The distinguishing
characteristics and assemblages that had been obtained
in this study can serve as valuable information in the
identification of dematiaceous-endophytic fungi that are
present in other plants. To further explore their possible
role in the ecology of barnyard grass, however, it is
best recommended that their abundance, diversity, and
dominance in tissues of the weed be studied.
ACKNOWLEDGEMENTS
Special thanks to the Department of Agriculture,
Philippine Rice Research Institute, Department of
Science and Technology-Science Education Institute, and
Philippine Council for Agriculture, Aquatic and Natural
Resources Research and Development for the thesis
support granted to the first author.
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