Microbes Environ. Vol. 26, No. 3, 228–236, 2011 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10163

Fungal Assemblages in the Rhizosphere and Rhizoplane of Grasses of the Subfamily Panicoideae in the Lakkavalli Region of Karnataka, India

MADHUGIRI MALLAIAH VASANTHAKUMARI1, and MANCHANAHALLY BYRAPPA SHIVANNA1* 1Department of P.G. Studies and Research in Applied Botany, Kuvempu University, Shankaraghatta 577451, Shimoga District, Karnataka, India (Received September 3, 2010—Accepted April 24, 2011—Published online June 11, 2011)

Fungal communities associated with roots play an important role in nutrient cycling, supporting plant growth and the biocontrol of plant diseases. Experiments were conducted in 2004–2006 to isolate and characterize, based on their morphological features, rhizosphere and rhizoplane fungi from perennial grasses of the subfamily Panicoideae growing in forests of the Western Ghats in India. Fungal species were isolated on potato dextrose, czapeck dox and water agar, in different locations and seasons. The results obtained on PDA were used for detailed analysis since most fungi occurred in high percentages. While certain grasses harbored diverse fungal species, others supported only a few species. Most fungi were isolated during winter followed by the rainy and summer seasons. The species richness, diversity and evenness of fungal assemblages in the rhizosphere and rhizoplane depended on the grass species and season. Ascomycetes were isolated in large numbers in most grass species. Species of Aspergillus, Chaetomium, and Trichoderma occurred frequently. Certain others and non-sporulating fungi were grass species-specific. Most fungal species colonized the middle of the root more than the root tip or root base. Results suggest that perennial grasses harbor diverse fungal communities whose potential could be tapped for producing secondary metabolites and managing plant diseases. Key words: Fungal diversity, Western Ghats, perennial grass, seasonal variation, Ascomycetes

Fungi colonize diverse habitats and substrates and play a 37, 40, 45). These studies prompted the authors to document substantial role in plant health and productivity as well as in fungal assemblages in the rhizosphere of perennial grasses disease. The rhizosphere is the zone of soil directly influenced of India and evaluate their biocontrol characteristics. Based by the roots of plants and provides a distinct biologically on this, a survey of rhizosphere fungi in certain perennial active micro-habitat within the terrestrial ecosystem (5, 11, grass species was undertaken which indicated the occurrence 30, 41). The organic compounds present in root exudates of anamorphic and teleomorphic ascomycetes, anamorphic make the rhizosphere environment very different from that basidiomycetes, zygomycetes and certain non-sporulating of the bulk soil and support a hoard of saprophytic fungi (Vasanthakumari et al. 2006; conference abstract). In microorganisms. These microorganisms are attracted to the this context, the isolation and characterization of rhizosphere release of ions, oxygen and water, in addition to carbon- and rhizoplane fungi from certain perennial grass species of containing compounds, amino acids, hormones and vitamins the subfamily Panicoideae were taken up. An attempt was (52). There is a considerable body of evidence which suggests also made to document variation in the diversity of fungal that the loss of soluble organic substances from roots is communities in relation to season, growing stage and location. significantly stimulated by the presence of microbes (33, 55). The present study is aimed at understanding the diversity, A search of the literature indicated that certain plant species evenness and richness of fungal communities in different sea- have been studied extensively for the occurrence of fungi in sons in the rhizosphere and rhizoplane of certain Panicoideae the rhizosphere and rhizoplane (34). A number of fungi have grasses growing in the area adjoining the Bhadra reservoir in been isolated and characterized from the rhizosphere of crops the Lakkavalli state forest of Karnataka, India. and to some extent, non-crop plants (1, 2, 29, 39). However, few reports are available on the occurrence of rhizosphere Materials and Methods fungi in grass species (23, 26, 42, 46). The diversity of rhizosphere fungi has been shown to vary depending on Study site season, location and soil type (7). Fungal species have been The study was carried out in the Bhadra Wildlife sanctuary, characterized mainly on a morphological basis; however, located in the Western Ghats region of Karnataka, India. The Western Ghats is recognized as a ‘hot spot’ of global biodiversity. some attempts have been made using molecular techniques The Lakkavalli state forest (13°34'–13°39' N latitude, 75°36'–75°39' (23, 42). Previous work on the grass rhizosphere indicated E longitude), an area adjoining the Bhadra reservoir situated in the the occurrence of a large number of fungal species, of which peripheral region of the Bhadra Wildlife sanctuary, was selected as a few were studied for their growth promotion and biocontrol the study area. Three study sites were established at a distance of potential and systemic resistance inducing ability (15, 27, 36, two kilometers from each other within the study area. Three quadrates (1×1 m) representing three replicates were established at each site. The data were collected in July, November and March, * Corresponding author. E-mail: [email protected]; representing the rainy, winter and summer seasons, of 2004–2006. Tel: +91–9448836859; Fax: +91–08282–256255. Fungal Assemblages in Panicoideae Grasses 229

Plant species and sampling design design (RCBD) and trials were conducted during 2004–05 and 2005– Five perennial grass species—Alloteropsis cimicina (L.) Stapf. 06. Fungal populations from the rhizosphere samples were quantified −1 (Bug seed-grass), Digitaria bicornis (Lam). Roem. & Schult as the number of colony forming units (cfu g ). The root colonization Beauv. (Asian crab grass), Heteropogon contortus (L.) P. Beauv. frequency (%) of rhizoplane fungi (20) and the predominantly (Tangle head), Ischaemum ciliare Retz. (Indian murianagrass) and occurring fungi (%) (31) were calculated. The diversity, evenness Paspalidium flavidum (Retz.) A. Camus (Shot grass) growing in the and richness of fungal species in the rhizosphere and rhizoplane of study area were selected. Initially, all species were identified to the grass species were analyzed using Simpson and Shannon indices. tribe level in the field based on vegetative and floral characteristics Jaccard’s similarity co-efficient and rarefaction indices were also (8) and characterized to the species level as per the descriptions in calculated (http://palaeo-electronica.org/2001. Accessed 20 Decem- manuals (10, 47, 57). The voucher specimens were collected at ber 2008). different growth stages. The identity of grasses was kindly confirmed by a grass specialist, Dr. G.K. Bhat, Retired Professor, Poornaprajna Results College, Udupi, Karnataka. Simultaneously, herbarium specimens were also prepared and deposited in the Department of Applied Study area, soil mineral nutrients and grass species Botany for future reference. Tests of homogeneity of two trials by ANOVA indicated Collection of soil samples no significant difference in the incidence of fungal commu- Soil samples were collected from soil adjacent to grass plants of nities isolated from rhizospheres and rhizoplanes of three each species up to a depth of 15–20 cm. Soil samples from each quadrate were pooled, air-dried and tested regarding soil pH, organic locations in the study area. Therefore, the data for the two carbon and available N, P and K (4, 28, 48). trials over three locations were averaged and subjected to ANOVAs and further analyses. Preparation of rhizosphere and rhizoplane samples The mineral nutrient composition of bulk soil (pH 5.3– Rhizosphere and rhizoplane samples were collected from five 5.6) collected from different locations did not vary much. plants (per quadrate) of each grass species. The zone of soil immediately adjacent to the roots of each plant (2 cm from the However, the N, P and K contents of soil samples varied in −1 culm) up to a depth of 15–20 cm was removed carefully using a different seasons. The N content was high (4.05 kg ha each) trowel. Samples consisting of soil as well as root systems were during winter and summer and very low (0.36 kg ha−1) during collected and brought to the laboratory in separate sterile polypro- the rainy season. However, the P and K contents were highest pylene bags and used within 4 h of collection. The root systems during winter (23.05 and 10.12 kg ha−1, respectively) followed were carefully removed from the sample and gently shaken off to by the rainy season (16.59 and 9.51 kg ha−1, respectively). remove superfluous soil. Soil particles closely adhering to the roots In summer, their availability was low (15.98 and 7.69 kg were collected by gentle scraping using a sterile spatula and brushing −1 with a camel hair brush. The soil so collected formed the rhizosphere ha , respectively). The organic carbon content remained the sample. The root system without soil particles was considered the same (0.99–1.00%). rhizoplane sample. Out of 20 perennial grass species in the study area, five Isolation and characterization of fungal species from the species of the sub family Panicoideae were selected for the rhizosphere and rhizoplane study as they were abundantly available through different sea- Soil dilution plating (dilution-10−4) of rhizosphere samples was sons. Ischaemum ciliare, Digitaria bicornis and Heteropogon done on streptomycin (100 mg L−1)-amended potato dextrose agar, contortus flowered profusely during November-February, czapek dox or water agar (1%) media (PDA and CZA, Himedia, Paspalidium flavidum flowered in August-September, and Mumbai) (16). The plates were incubated in a chamber under 12/ Alloteropsis cimicina flowered throughout the year. The study 12 h cycles of light and darkness at 23±2°C for five to six days. area supported herbaceous plants and shrubs, and a few tree Root samples of each grass species (100 roots/species) were species. washed in slow running tap water for 30 min, and then in sterile distilled water twice, and the excess water blotted off with sterile Fungal occurrence and composition of media blotter discs. The root along the axis was divided in to a base, middle and tip and each region was fragmented into 1-cm long Fungal species have been characterized based on morpho- segments (36). Root segments were placed on 9-cm dia. Petri dishes logical markers like colony habit and color, fruiting bodies, containing the above media amended with streptomycin (100 mg −1 color, size, shape and septation in spores. The identification L ) and incubated as described earlier. of certain species was confirmed by two mycologists; Prof. The fungal species were characterized based on their cultural characteristics and the morphology of fruiting bodies and conidia/ Emeritus Dr. C. Manoharachary, Osmania University, Hyder- spores using manuals (3, 6, 9, 17, 19, 49, 50). The species abad and Prof. D. J. Bhat, Goa University, Goa. In comparison nomenclature was confirmed by visiting Index Fungorum to other media, PDA supported a large number of fungal (www.indexfungorum.org). Further, cultures in different media were isolates. Most isolates were common to both PDA and CZA, also subjected to near-UV (366 nm) light treatment for 5–6 days. however, Ambrosiella xylebori Brader ex Arx & Hennebert, The fungal isolates failing to produce reproductive propagules on Arthrobotrys conoides Drechsler, Humicola grisea Traaen., PDA/CZA were sub-cultured on water agar (1%) or malt extract Scopulariopsis brumptii Salv.-Duval, and Scolecobasidium agar (Himedia, Mumbai). The cultures failing to sporulate on these media were considered non-sporulating fungi (NSF). The NSF were tshawytschae (Doty & D.W. Slater) McGinnis & Ajello were tested for clamp connections or crosiers, for grouping under exclusively observed on CZA. Likewise, Amorphotheca ascomycetes or basidiomycetes. Voucher numbers were assigned to resinae Parbery, Arthrobotrys conoides Fresen, Beltrania all the fungal isolates from rhizosphere and rhizoplane regions, rhombica Penz, Mammaria echinobotryoides Ces, Stachy- documented and deposited in the Department of Applied Botany, botrys chartarum (Ehrenb.) S. Hughes, Pestalotiopsis Kuvempu University. disseminata (Thüm.) Steyaert and Mucor hiemalis Wehmer Analyses of data were observed only on PDA. On the other hand, water agar The experimental arrangement was a randomized complete block supported pycnidia-producing fungi. Hence, only the data 230 VASANTHAKUMARI et al. pertaining to PDA was analyzed and described in this paper. isolated in considerable numbers during winter followed by The NSF failed to produce fruiting bodies on MEA or upon the rainy season. However, a few fungal communities UV light treatment and had septate mycelia (aerial cottony appeared during summer (Table 1). or submerged) with colored or hyaline hyphae, and lacked Fungal isolates were grouped into zygomycota, (4.78 clamp connections or crosiers. %), anamorphic (78.14%) and teleomorphic (10.66%) , anamorphic basidiomycota (0.29%), and non- Fungal communities in the rhizosphere sporulating fungal (NSF) isolates (6.09%) (Fig. S1). Results of dilution plating indicated that the rhizosphere Aspergillus, Alternaria, Cladosporium, Fusarium (F. of five perennial grass species harbored 704.44 cfu of fungal oxysporum), Khuskia, Myrothecium, Penicillium, Pesta- species per gram of soil on PDA. The fungal isolates belonged lotiopsis, Trichoderma, and Macrophomina occurred most to 54 species of 31 genera. Ischaemum ciliare ranked the top frequently. Among the above, species of Aspergillus, Peni- of the list, hosting 190.98 cfu, followed by Digitaria bicornis cillium, and Trichoderma occurred in high frequency (Table (132.29 cfu), while Alloteropsis cimicina was the poor 2). Chaetomium globosum, and supporter of fungal isolates. Fungal communities were Plagiostoma sp. (teleomorphic ascomycetes), and Cunning-

Table 1. Occurrence of fungal species in the rhizosphere and rhizoplane of grass species of the subfamily Panicoideae on PDA during different seasons1

Rhizosphere (cfu g−1 soil) Rhizoplane (% colonization frequency) Grass species Rainy2 Winter2 Summer2 Rainy Winter Summer Alloteropsis cimicina 41.3 46.0 38.0 436.0 573.0 347.0 Digitaria bicornis 45.0 58.6 28.6 526.0 633.0 444.0 Heteropogon contortus 39.3 56.9 34.6 453.0 616.0 378.0 Ischaemum ciliare 56.0 82.6 52.3 474.0 856.0 353.0 Paspalidium flavidum 42.2 49.6 38.0 424.0 577.0 275.0 1Data is based on the values of three study sites and three seasons of two trials. 2Rainy, winter and summer seasons are represented by July, November and March of 2004–2006; LSD for comparing treatment means of rhizosphere fungi is 0.029 (P=0.01) and 0.038 (P=0.001) and rhizoplane fungi is 0.047 (P=0.01) and 0.062 (P=0.001) among seasons or grass species.

Table 2. Frequency of anamorphic ascomycetes isolated from the rhizosphere of grass species of the subfamily Panicoideae on PDA1 Frequency of occurrence2 (%) Sl. No. Fungal species A. cimicina D. bicornis H. contortus I. ciliare P. flavidum 1 Acrophialophora fusispora (S.B. Saksena) Samson W. Gams 2.123 —4 ——— 2 Aspergillus spp.5 20.21 (3)6 16.12 (3) 14.69 (2) 17.83 (3) 17.26 (2) 3 Botryotrichum piluliferum Sacc. & Marchal 2.66 — — — — 4 Cladosporium spp.7 7.18 (1) 5.54 (1) 11.9 (2) 15.08 (3) 13.79 (2) 5 Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams — — — 6.00 8.18 6 Colletotrichum dematium (Pers.) Grove 2.39 — — — — 7 Fusarium oxysporum E.F. Sm. & Swingle 6.65 5.03 6.33 6.86 8.18 8 Macrophomina phaseolina (Tassi) Goid. — 2.52 1.26 — — 9 Myrothecium roridum Tode 3.45 2.27 2.78 2.22 2.40 10 Khuskia oryzae H.J. Huds 4.79 4.62 4.81 4.11 4.00 11 Penicillium spp.8 7.71 (1) 9.57 (2) 21.52 (3) 17.82 (3) 15.39 (2) 12 Pestalotiopsis spp.9 3.46 (1) 1.00 (1) 2.53 (1) — 3.20 (1) 13 Trichoderma spp.10 9.30 (2) 14.10 (2) 7.60 (1) 7.54 (2) 6.76 (1) 14 Verticillium albo-atrum Reinke & Berthold — — — 2.05 — Total frequency 71.77 (16) 63.28 (14) 76.45 (16) 82.74 (20) 83.42 (15) 1Data is based on the values for three study sites and three seasons in two trials; 2Frequency of fungal occurrence was calculated based on the colony forming units of a particular over the sum of colony forming units and is represented as a percentage; 3Data are an average of three replicates, each with 81 samples; 4Not observed; 5Aspergillus species=A. candidus Link (0–1.51), A. flavus Link. (7.05–8.45), A. nidulans (Eidam) G. Winter. (0–7.71), A. brasiliensis Varga, Frisvad & Samson. (4.52–8.81), A. terreus Thom. (0–1.88); 6Figures in parentheses indicate total number of species of the genera which may vary in different grasses; 7Cladosporium species=C. cladosporioides (Fresen.) G.A. de Vries. (5.54–7.92), C. herbarum (Pers.) Link. (5.87–7.20), C. oxysporum Berk. & M.A. Curtis. (0–1.71); 8Penicillium species=P. chrysogenum Thom. (7.09–7.54), P. citrinum Sopp. (6.34–8.10), P. islandicum Sopp (0–7.71), P. oxalicum Currie & Thom. (3.94–6.98), P. rubrum Sopp. (0–2.27); 9Pestalotiopsis species=P. disseminata (Thüm.) Steyaert. (0–1.00), P. glandicola (Castagne) Steyaert. (3.20–3.46), P. guepini (Desm.) Steyaert. (0–2.53); 10Trichoderma species = T. asperellum Samuels, Lieckf. & Nirenberg.(0–1.86), T. harzianum Rifai. (5.83–7.44), T koningii Oudem. (0–7.05), T. viride Pers. (0–1.7). Anamorphic ascomycetes-Gliomastix cerealis (P. Karst.) C.H. Dickinson, Alternaria species=A. alternata (Fr.) Keissl., A. tenuissima (Kunze) Wiltshire, Arthrobotrys oligospora Fresen. Beltrania rhombica Penz., inaequalis (Shear) Boedijn. Epicoccum nigrum Link, Mammaria echinobotryoides Ces., Oidiodendron Robak, Phomopsis vexans (Sacc. & P. Syd.) Harter, Stachybotrys chartarum (Ehrenb.) S. Hughes and Trichothecium roseum (Pers.) Link. with <2% frequency are also included in the total frequency of respective grass species. Fungal Assemblages in Panicoideae Grasses 231

Table 3. Frequency of fungal species grouped under teleomorphic ascomycetes, anamorphic basidiomycetes, zygomycota and non-sporulating fungi isolated from the rhizosphere of grass species of the subfamily Panicoideae on PDA1 Frequency of occurrence2 (%) Sl. No. Fungal species/isolates A. cimicina D. bicornis H. contortus I. ciliare P. flavidum Teleomorphic ascomycetes 1 Chaetomium spp.3 —4 5.29 (1)5 2.02 (1) 2.91 (1) 2.13 (2) 2 Cochliobolus lunatus R.R. Nelson & F.A. Haasis 7.186 5.29 3.54 4.11 4.89 3 Gibberella fujikuroi (Sawada) Wollenw 7.71 2.51 — — — 4 Haematonectria haematococca (Berk. & Broome) —2.52——— Samuels & Rossman 5 Plagiostoma sp. 3.45 — 2.78 (1) — — Total frequency 18.34 (3) 15.61 (4) 9.35 (4) 7.02 (2) 7.02 (3) Anamorphic basidiomycetes 1 Trichosporon beigelii (Küchenm. & Rabenh.) Vuill — — 1.77 — — Total frequency — — 1.77 (1) — — Zygomycota 1 Cunninghamella spp. 5.31 (2) 4.28 (1) 5.32 (1) 4.97 (1) 3.20 (2) Total frequency 5.31 (2) 4.28 (1) 5.32 (1) 4.97 (1) 3.81 (3) Non-sporulating fungi 1 NSF isolate 51 2.67 — — — — 2 NSF isolate 129 — 2.01 — — — 3 NSF isolate 132 — 2.17 — — — 4 NSF isolate DB27 — — — — — 5 NSF isolate 196 — — 2.78 — — 6 NSF isolate 168 — — — — 2.93 Total frequency 4.53 (2) 5.94 (3) 6.32 (3) 5.11 (4) 5.68 (3) 1Data is based on the values for three study sites and three seasons in two trials; 2Frequency of fungal occurrence was calculated based on the colony forming units of a particular fungus over the sum of colony forming units and is represented as a percentage; 3Chaetomium species=C. elatum Kunze.(0–0.80), C. globosum Kunze. (1.33–5.29); 4Not observed; 5Figures in parentheses indicate the total number of species of the genera which may vary in different grasses; 6Data are the average of three replicates, each with 81 samples. Teleomorphic ascomycetes—Amorphotheca resinae Parbery, zygomycota (Cunninghamella species=C. echinulata (Thaxt.) Thaxt. (3.20–5.32), C. elegans Lendn. (0–1.59), Mucor hiemalis Wehmer); and NSF (isolates 9, 127, DB27, 191,193, IC 24, 118, 147, 82, 178, 114) with <2% frequency are included in the total frequency of respective grass species.

Table 4. Species richness, diversity and evenness indices of fungal communities in the rhizosphere and rhizoplane of grasses of the subfamily Panicoideae on PDA1 Diversity index Evenness index Root regions/grass species Species richness Shannon (H’) Simpson (D’) Shannon (J’) Simpson (E’) Rhizosphere2 Alloteropsis cimicina 23.0 2.96 17.42 0.94 0.75 Digitaria bicornis 27.0 2.87 20.29 0.87 0.75 Heteropogon contortus 26.0 3.03 18.31 0.93 0.70 Ischaemum ciliare 27.0 3.05 18.35 0.92 0.68 Paspalidium flavidum 23.0 2.89 15.79 0.92 0.68 Rhizoplane3 Alloteropsis cimicina 24.0 2.99 17.73 0.94 0.74 Digitaria bicornis 28.0 3.14 20.63 0.94 0.73 Heteropogon contortus 26.0 3.21 19.24 0.98 0.72 Ischaemum ciliare 28.0 3.07 18.73 0.92 0.66 Paspalidium flavidum 24.0 2.95 17.17 0.92 0.72 1Data based on the values for three study sites and three seasons in two trials; 2Data are the average of three replicates, each with 81 samples; 3300 root segments/grass species. hamella echinulata (zygomycota) occurred in various per- not during the rainy season (Fig. S1). centages (Table 3). Among the NSF, two from Alloteropsis The species richness of the fungal community ranged from cimicina, three each from Digitaria bicornis, Heteropogon 23 to 27 on PDA and was high in D. bicornis and I. ciliare. contortus, and Paspalidium flavidum, and four from The Shannon diversity index (H’) of the rhizosphere fungal Ischaemum ciliare were isolated (Table 3). The rainy season community was highest (H’=3.05) for I. ciliare and H. favored maximum expression of anamorphic ascomycetous contortus (H’=3.03). The Shannon evenness (J’) index was fungi, followed by winter and summer. The NSF numbers highest for A. cimicina (J’=0.94) and H. contortus (J’=0.93), were considerable during summer followed by winter, but followed by I. ciliare and P. flavidum. The Simpson diversity 232 VASANTHAKUMARI et al.

Table 5. Jaccard’s similarity co-efficient1 of fungal communities in the rhizosphere and rhizoplane of grass species of the subfamily Panicoideae during different seasons2 Rhizosphere3 Rhizoplane4 Grass species Rainy5 Winter5 Summer5 Rainy Winter Summer Alloteropsis cimicina 0.78 0.97 0.75 0.78 0.81 0.66 Digitaria bicornis 0.82 0.65 0.52 0.82 0.71 0.51 Heteropogon contortus 0.93 0.59 0.53 0.73 0.74 0.56 Ischaemum ciliare 0.84 0.79 0.84 0.85 0.66 0.68 Paspalidium flavidum 0.74 0.78 0.66 0.66 0.81 0.68 1Similarity co-efficient ranges from 0 for complete dissimilarity to 1.00 for complete similarity; 2Data based on the values to three study sites and three seasons in two trials; 3Data are the average of three replicates, each with 81 samples; 4Data are the average of three replicates each with 300 root segments/grass species; 5Rainy, winter and summer seasons are represented by July, November and March of 2004–2006.

Table 6. Frequency of anamorphic ascomycetes isolated from the rhizoplane of grasses of the subfamily Panicoideae on PDA1 Frequency of occurrence2 (%) Sl. No. Fungal species/isolates Alloteropsis Digitaria Heteropogon Ischaemum Paspalidium cimicina bicornis contortus ciliare flavidum 1 Acrophialophora fusispora 2.583 —4 ——— 2 Aspergillus spp.5 21.89 (4)6 14.89 (3) 11.54 (2) 15.5 (3) 14.34 (2) 3 Cladosporium spp.7 7.77 (2) 9.35 (2) 9.36 (2) 13.48 (2) 12.14 (2) 4 Fusarium oxysporum E.F. Sm. & Swingle 6.19 4.93 6.97 6.83 7.52 5 Clonostachys rosea (Link) Schroers — — — 7.13 7.05 6 Myrothecium roridum Tode 2.87 2.43 3.11 1.72 2.89 7 Khuskia oryzae H.J. Huds 5.60 5.30 5.25 3.62 4.94 8 Penicillium spp.8 8.25 (1) 9.67 (2) 21.49 (3) 17.00 (3) 15.75 (2) 9 Pestalotiopsis spp.9 2.80 (1) 0.93 (1) 2.43 (1) 0.55 (1) 4.15 (1) 10 Trichoderma spp.10 10.24 (2) 11.15 (2) 7.67 (1) 9.92 (2) 6.89 (1) Total frequency 73.42 (18) 65.14 (16) 75.38 (18) 81.50 (19) 78.79 (15) 1Data based on the values for three study sites and three seasons in two trials; 2The colonization frequency of each fungus was calculated based on the number of root segments colonized by a fungus over the total number of segments assessed and is represented as a percentage; 3Data are the average of three replicates, each with 81 samples; 4Not observed; 5Aspergillus species=A. candidus (0–1.31), A. flavus (6.77–7.68), A. nidulans (0–6.34), A. brasiliensis (4.00–7.30), A. tamarii Kita. (0–0.66), A. terreus (0–2.14); 6Figures in parentheses indicate the total number of species of the genera which may vary in different grasses; 7Cladosporium species=C. cladosporioides (4.86–6.47), C. herbarum (4.49–7.01), C. oxysporum (0–2.02); 8Penicillium species=P. chrysogenum (7.07–7.91), P. citrinum (6.77–7.84), P. islandicum (0–8.24), P. oxalicum (3.90–7.46), P. rubrum (0–2.56); 9Pestalotiopsis species=P. disseminata (0–0.93), P. glandicola (0–0.93), P. guepini (0.55–2.43); 10Trichoderma species=T. asperellum (0–1.76), T. harzianum (6.89–8.48), T. koningii (0–6.79), T. viride (0–2.61). Anamorphic ascomycota—Aureobasidium pullulans (de Bary) G. Arnaud., Alternaria species=A. alternata (Fr.) Keissl., A. tenuissima (Kunze) Wiltshire, Amorphotheca resinae, Arthrobotrys oligospora Fresen, Beltrania rhombica, Botryotrichum piluliferum, Colletotrichum dematium, Curvularia inaequalis (Shear) Boedijn. Epicoccum nigrum Link, Mammaria echinobotryoides Ces., Macrophomina phaseolina, Gliomastix cerealis (P. Karst.), Oidiodendron griseum Robak, Phomopsis vexans (Sacc. & P. Syd.), Stachybotrys chartarum, Trichothecium roseum and Verticillium albo-atrum with <2% frequency are included in the total fre- quency of respective grass species. index (D’) was highest for D. bicornis (D’=20.29) followed characterized into 57 species of 34 genera. They were grouped by I. ciliare and H. contortus. The Simpson evenness index into anamorphic ascomycota (75.91%), teleomorphic asco- (E’) was highest for A. cimicina and D. bicornis (Table 4). mycota (10.23%), anamorphic basidiomycota (0.35%), The Jaccard’s similarity co-efficient showed moderate to high zygomycota (6.28%), and NSF (7.20%) (Fig. S1). The similarity of assemblages of fungal communities (0.52 to rhizoplane grasses were predominantly colonized by 0.97) in different grass species and seasons. High similarity anamorphic and teleomorphic ascomycetous fungi during co-efficients were obtained for A. cimicina, H. contortus and the rainy season followed by winter and summer. On the I. ciliare during the winter, rainy and summer seasons, other hand, zygomycota and NSF were encountered more respectively (Table 5). The rarefaction index indicated that often in summer and winter than the rainy season. Species the expected number of fungal species always increased with of Aspergillus, Cladosporium, Curvularia (C. inaequalis), the increase in the number of isolates from the rhizosphere Fusarium (F. oxysporum), Myrothecium, Pestalotiopsis, of grass species. The number of isolates was highest during Trichoderma, and Macrophomina phaseolina colonized the winter followed by the rainy season and summer in all the rhizoplane of most grasses to a maximum extent (Table 6). grass species (Fig. S2). Penicillium islandicum and Trichoderma harzianum fre- quently occurred in A. cimicina (Table 6). Chaetomium Fungal communities in the rhizoplane globosum and Cunninghamella echinulata were isolated The root segments of all grass species yielded a total of from root segments of most grass species (Table 7), while 7,365 isolates of fungi (Table 1) on PDA, which were Plagiostoma sp. was isolated from at least two grass species. Fungal Assemblages in Panicoideae Grasses 233

Table 7. Frequency of fungal species grouped under teleomorphic ascomycetes, anamorphic basidiomycetes, zygomycota and non-sporulating fungi isolated from the rhizoplane of grass species of the subfamily Panicoideae on PDA1 Frequency of occurrence2 (%) Sl. No. Fungal species/isolates Alloteropsis Digitaria Heteropogon Ischaemum Paspalidium cimicina bicornis contortus ciliare flavidum Teleomorphic ascomycetes 1 Chaetomium spp.3 —4 4.68 (1)5 1.59 (1) 1.66 (1) 3.05 (2) 2 Cochliobolus spp.6 4.64 (1) 6.30 (2) 3.18 (1) 7.01 (2) 5.25 (1) 3 Gibberella fujikuroi (Sawada) Wollenw 7.08 2.18 — — — 4 Haematonectria haematococca (Berk. & Broome) —2.37——— Samuels & Rossman 5 Plagiostoma sp. 3.46 (1) — 3.11 (1) — — Total frequency 15.18 (3) 15.53 (5) 7.88 (3) 7.01 (2) 8.30 (3) Anamorphic basidiomycetes 1 Trichosporon beigelii ——1.94—— Total frequency — — 1.94 — — Zygomycota 1 Cunninghamella spp.7 7.74 (2) 5.24 (1) 5.53 (1) 3.03 (1) 4.94 (1) 2 Mucor hiemalis ————2.74 (1) Total frequency 7.74 (2) 5.24 5.53 4.09 (2) 7.68 (2) Non-sporulating fungi (NSF) 1 Isolate 9 2.87 — — — — 2 Isolate 51 2.72 — — — — 3 Isolate 129 — 2.62 — — — 4 Isolate 132 — 3.30 — — — 5 Isolate 136 — 3.68 — — — 6 Isolate 193 — — 2.07 — — 7 Isolate 196 — — 2.35 — — 8Isolate 179 ————2.43 Total frequency 5.59 (2) 13.14 (6) 5.73 (3) 5.33 (4) 5.17 (4) 1Data are based on the values for three study sites and three seasons in two trials. Data are the average of three replicates, each with 81 samples; 2The colonization frequency of each fungus was calculated based on the number of root segments colonized by a fungus over the total number of segments assessed and is represented as a percentage; 3Chaetomium species=C. elatum (0–0.94), C. globosum (1.59–4.68); 4Not observed; 5Figures in parentheses indicate total number of species of the genera which may vary in different grasses; 6Cochliobolus species = C. lunatus (3.18–5.35), C. spicifer R.R. Nelson (0–1.37); 7Cunninghamella species = C. echinulata (3.03–5.68), C. elegans (0–2.06). Teleomorphic ascomycetes—Gliomastix cerealis (P. Karst.), species of zygomycota (Absidia glauca Hagem) and NSF (isolates 127, 139, DB27, 191, IC24, 118, 147, 82, 178, 168) with <2% frequency are included in the total frequency of respective grass species.

The NSF had specific affinity for roots of certain grass (including NSF, 323 isolates), closely followed by the root species; 2, 3, 4, 4 and 6 isolates colonized roots of A. cimicina, tip (258 isolates) and root base (222 isolates) (Table S1). H. contortus, I. ciliare, P. flavidum and D. bicornis, Aspergillus flavus, A. brasiliensis, C. globosum, C. respectively (Table 7). cladosporioides, Cunninghamella echinulata, Cochliobolus The species richness of rhizoplane fungal communities lunatus, Fusarium oxysporum, Myrothecium roridum, ranged between 24 and 28 and was high in D. bicornis and Khuskia oryzae, Penicillium chrysogenum and Trichoderma I. ciliare. High Shannon (H’) and Simpson (D’) diversity harzianum colonized all regions of the root in most grass indices were observed for H. contortus (H’=3.21; D’=19.24) species. In contrast, isolates that colonized exclusively certain and D. bicornis (H’=3.14; D’=20.63). The Simpson evenness regions of grass roots include Colletotrichum dematium, index was highest for A. cimicina (E’=0.74) and D. bicornis Penicillium islandicum and Trichoderma asperellum and 2 (E’=0.73). Heteropogon contortus (J’=0.98) had the highest NSF from A. cimicina; A. candidus, Cochliobolus spicifer, Shannon evenness index (J’) value (Table 4). The Jaccard’s Fusarium solani (Haematonectria haematocca), Penicillium similarity co-efficient of fungal communities from the rubrum, Pestalotiopsis disseminata, Phomopsis vexans, T. rhizoplane ranged from 0.51 to 0.85, with high values for D. koningii and 6 NSF from D. bicornis; Alternaria alternata, bicornis and I. ciliare during the rainy season (Table 5). In Trichothecium roseum and 3 NSF from H. contortus; A. the rhizoplane, the expected number of species (ES) increased tenuissima, A. terreus, Cladosporium oxysporum, Curvularia with the number of isolates from all regions of roots in grass inaequalis, Verticillium albo-atrum and 4 NSF from I. species; however, only up to 100 isolates, after which it ciliare, and Gliomastix cerealis, Epicoccum nigrum and 4 remained the same irrespective of season or root region (Fig. NSF from P. flavidum (Table S1). S2). The root colonization ability of fungal communities Discussion depended on the grass species and season. The mid root region of the host was preferred by most fungal species Results of the two trials were similar, perhaps due to similar 234 VASANTHAKUMARI et al. agroclimatic conditions at all three locations. The study species failing to sporulate on culture media could be involved the identification of more than 7,000 fungal isolates; characterized by molecular techniques involving the ITS of hence molecular techniques could not be employed. rRNA. Molecular techniques have successfully been used to characterize fungi (42, 43, 53). Interesting observations Fungal communities in response to soil nutrients and include the grass-specific affinity of NSF and predomination seasonal variation of anamorphic and teleomorphic ascomycetes in the rhizo- The availability of nutrients in the soil from the three sphere and colonization of the entire root in all grass species. locations did not vary much because of similar micro- Extensive colonization of the middle of the root by fungal ecological conditions, which might have favored similar communities might depend upon the root exudate produced fungal communities. The soil nutrient content was high during and fungal root competence. In cucumber, the root base winter, since the preceding rainy season solubilized minerals supported extensive fungal colonization (36). In some, the available in soil layers. Optimum water potential is shown zone of elongation behind the root tip supported the growth to enhance soil nutrient availability thus favoring microbial of primary root colonizers (56). The selective availability of growth (14, 44). a carbon source and organic acids might determine fungal The flowering in I. ciliare, D. bicornis and H. contortus communities in different regions of the root. during the winter co-incided with the species richness of Among the dominant ascomycetous fungi, certain species fungal communities. On the other hand, the flowering in P. were frequently isolated from the rhizosphere of plants as flavidum at the end of the rainy season co-incided with the well as from bulk soil (34). Species of Penicillium and rich fungal community during winter. This suggested the Aspergillus documented from other soil types (17) were also availability of mineral nutrients in soil to play a crucial role recorded along with species of Chaetomium, Cochliobolus, in the occurrence of fungal communities in the rhizosphere Fusarium, Myrothecium, Plagiostoma and Trichoderma and rhizoplane. Additionally, the fibrous root system of grass (Tables 3 and 7). Species of Trichoderma (29) and certain species during winter might increase the root exudate content NSF (34) from the rhizosphere and rhizoplane of plants (29) in soil. This exudate was shown to promote fungal commu- have been tested for biocontrol. nities in the rhizosphere and rhizoplane (12) and at rhizo- Jaccard’s similarity co-efficient analysis indicated that sphere deposits during various plant growth stages (25). Our fungal assemblages, rather than species richness, differed results corroborated the findings of Marschner et al. (35) and among seasons. However, in certain grass species, the Dunfield and Germida (18). variation in fungal assemblages was marginal, although the population of some communities was high. This suggested Rhizosphere and rhizoplane fungal community structure that a wide range of fungal communities occur in certain Information on changes in the fungal community structure grasses, while specific fungal communities occurred in others. in the rhizosphere and rhizoplane of grasses is very limited. Variations in the composition of fungal assemblages between Certain grasses support a large number of fungal communities two study sites are reported (51, 54). The increase in the in the rhizosphere and rhizoplane, while others do not. This expected number of species with the increase in number of suggested the host-specific selection of fungal communities isolates in most grass species suggested an abundance of in the rhizoplane which could be dependent on the root fungi in the rhizosphere and rhizoplane. This further exudate. The localization of fungal communities in the middle supported that high levels of root activity coupled with of the root might point out at their spatial distribution and exudate production provide shelter to many fungal species localization on the root surface. Earlier papers (22, 32, 42) in a particular season. also reported such root-fungal interactions. In conclusion, the two-year study indicated that five The enhanced isolation from the rhizosphere and perennial grass species of Panicoideae harbored a large rhizoplane of I. ciliare and D. bicornis during winter might number of fungal species in their rhizosphere and rhizoplane. suggest that fungal communities prefer a particular season Ascomycetous fungi occurred most often; the NSF was or grass species. This is reflected by the high richness and species-specific. High species richness coupled with moder- moderate to high diversity and evenness values. By contrast, ate to high diversity and evenness in certain grasses contrasted the two grass species with low richness, diversity and with low species diversity, richness and evenness in other evenness values might host specific fungal communities as grasses. The fungal assemblages were similar in most grass proposed by Broeckling et al. (12). The root morphology and species and the expected number of species increased along exudation pattern during plant development play a vital role with the number of isolates. Ascomycetous fungi with the in the variation and frequency of fungal communities in soil ability to colonize the middle of the root might have good in different seasons (13, 21, 38). Nutrient stress during root-colonizing capabilities. Certain predominant species of summer might support communities tolerant of water stress. fungi in the rhizosphere and rhizoplane of these grasses have Potato dextrose agar, rather than the other media, supported been studied in vitro for antagonistic activities and biocontrol a large number of fungal species suggesting its superiority potential (Vasanthakumari and Shivanna, unpublished data). as the isolation medium. The growth of certain fungal species In view of the failure of many biocontrol agents in the field only on CZA or PDA suggested the use of more than one (24), the identification and documentation of unknown and culture medium with a specific carbon source for the isolation novel fungal communities in rhizosphere soil require imme- of mycoflora from an ecological location. In the present study, diate attention. the authors consider that the morphological criteria are still necessary for identifying a fungal species. However, fungal Fungal Assemblages in Panicoideae Grasses 235

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