identities, characteristics, and assemblages of...

Philippine Journal of Science145 (2): 153-164, June 2016ISSN 0031 - 7683Date 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: [email protected] [email protected]

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).

INTRODUCTIONEndophytic 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

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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 METHODSSome 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.) KeisslerDescriptions 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

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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) WiltshireDescriptions 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).



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53' 54.6''. References: Barron (1968); Rayner (1970); Ellis (1971); Mew & Gonzales (2002); Pitt & Hocking (2009); Watanabe (2010).

Bipolaris australienses (M.B. Ellis) Tsuda. & UedaDescriptions 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-ds102Descriptions 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).



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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) ShoemakerDescriptions 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).



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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) BoedijnDescriptions 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. MathurDescriptions 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 BoedijnDescriptions 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-ds427Descriptions 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) PetchDescriptions 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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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).



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Stemphylium botryosum WallrothDescriptions 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-ds68-129Descriptions 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-ds392Descriptions 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).

DISCUSSIONDematiaceous 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.



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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.



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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].

CONCLUSIONThere 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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