summaries

A New Fungal Endophyte, Scolecobasidium humicola ,

Promotes Tomato Growth under Organic Nitrogen Conditions

Rola S. Mahmoud 1*

, Kazuhiko Narisawa 2

1 United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo, Japan, 2 College of Agriculture, Ibaraki

University, Ami machi, Ibaraki, Japan

Abstract

A new fungal endophyte, Scolecobasidium humicola , was identified as a common dark septate endophytic fungal

(DSE) species under both natural and agricultural conditions. This fungus was found to grow endophylically in the roots of tomato seedlings. Light microscopy of cross-sections of colonized tomato roots showed that the intercellular,

pigmented hyphae of the fungus were mostly limited to the epidermal layer and formed outer mantle-like structures. Two isolates of S. humicola, H2-2 and F1-3, have shown the ability to increase plant biomass with an organic

nitrogen source. This finding is the first report of S. humicola as an endophyte and could help to improve plant growth

with organic nitrogen sources.

Citation: Mahmoud RS, Narisawa K (2013) A New Fungal Endophyte, Scolecobasidium humicola, Promotes Tomato Growth under Organic Nitrogen

Conditions. PLoS ONE 8(11): e78746. doi:10.1371/journal.pone.0078746

Editor: Murad Ghanim, Volcani Center, Israel

Received June 19, 2013; Accepted September 21, 2013; Published November 1, 2013

permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 23380190) from the Japan Society for the Promotion of Science;

http://www.jsps.go.jp/english/e-grants/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

In the last few decades, interest in organic farming has

increased all over the world; however, only 0.9% of the world’s

agricultural lands are organic [1]. Organic farming practices aim to establish stable production systems with concern for nature,

in which the use of chemical nitrogen fertilizers, synthetic

pesticides and growth-promoting chemicals is not allowed [ 2].

The input of nutrients in organic fields generally relies on

organic fertilizers, manure, green manure, and/or crop residues; however, the balance of N mineralization/immobilization processes depends on the nature of these

substrates and on the ecological conditions of each agro-

system, which limit plant uptake and growth, especially at times

of peak crop demand [ 3]. Recently, an interesting finding about

plant nitrogen utilization in the forest ecosystem was reported

[4 ]. It was shown that the largest pool of N is typically organic,

e.g., amino acids and proteins are among the most abundant forms of organic N in the soil, comprising 80% of the soil N

supply, while ammonium and nitrate contribute only 10%. This

finding undoubtedly showed that symbiotic fungi, such as mycorhizal fungi, which can enhance host plant growth byimproving N nutrition, are extremely significant in the forest

ecosystem. This can be achieved via an increase in theabsorptive surface area provided by fungal hyphae, greater

uptake efficiency or by increasing access to various N sources

that are unavailable to non-mycorrhizal plants [ 5,6]. However

the ability of mycorrhizal fungi to maintain these benefits for

their host plant under some field conditions common in

industrialized agriculture is considered to be limited [7]. Dark septate endophytic fungi (DSE), which are a

miscellaneous group of ascomycetes colonize root tissues intracellularly and intercellularly without causing apparent negative effects on the host plant [ 8–10]. DSE associations

have been recognized in approx. 600 plant species of 320 genera in 114 families, including non-mycorrhizal plant species

[11 ]. DSE may benefit their host plants by facilitating the uptake

of plant mineral nutrients, including P, N and water [ 12–15],

and suppressing infection by plant pathogens [ 16–18]. Many

aspects of their ecological roles remain unclear although

several studies had focused on the abundance of DSE in different habitats. For that reason, the most suitable utilization

method of DSE for organic farming is still unclear. In our study, we hypothesized that natural and agriculture

systems might share similar DSE species, and DSE could play

a role in supporting plant growth under these agriculture systems, especially the organic farming system. In order to

prove this hypothesis, key DSE species were isolated andPLOS ONE | www.plosone.org1 November 2013 | Volume 8 | Issue 11 | e78746 identified in both natural and agriculture ecosystems. Here, we

describe the features or identity of the selected key DSE species that were effective in supporting tomato growth with

different organic nitrogen sources.

Materials and Methods Sample collection and fungal isolation Soil samples were collected in October 2010 from a forest,

organic field and conventional field at the Field Science Center of College of Agriculture, Ibaraki University Japan. Three soilsamples (approximately 300 ml) were collected at 0-20 cmdepth from each site and were kept in polyethylene bags and

stored at 4°C for a maximum of one month prior to utilization. Composite soil was prepared to bait endophytic fungi as

described by Narisawa et al. [ 17]. Each soil sample was

combined and mixed with autoclaved potting soil (Peat pot; Kureha Chemical Industry Co., Tokyo, Japan) at the ratio of 1:2 (v/v). The seeds of tomato Solanum lycopersicum cv. Gohobi

(Sakata Seed, Yokohama, Japan) and Chinese cabbage

Brassica campestris cv. Musou (Takii Seed, Kyoto, Japan)

were surface sterilized by immersion in a 70% solution of ethanol for 30 seconds, followed by a solution of sodium

hypochlorite (2% available chlorine) for 1.5 minutes. Then

seeds were rinsed three times with sterilized distillated water,

dried overnight and placed on 1.5% water agar medium (15 g Bacto agar [Difco Laboratories, Detroit, MI] for 1 liter distilled

water) in Petri dishes. After 4 days, the axenically grown seedlings were transplanted (three seedlings per pot) in to 90- mm diameter pots containing 100 ml composite soil. Each

collection site was considered as a bloc containing three replicates for each soil sample and plant species. Seedlings

were grown under greenhouse conditions with the temperature reaching 25°C. After three months, the roots collected from

young tomato and Chinese cabbage plants in each replicate were washed with running tap water to remove debris and cutinto approximately 1-cm segments. Fifteen root segments werechosen in random from each bait plant, washed three times in a 0.005% solution of Tween 20 (J.T. Baker Chemical Co.,Philipsburg, NJ), and rinsed three times with sterile distilled

water (SDW). The segments were air-dried overnight and

plated on nutrient agar containing 25 g. L -1

corn meal (infusion

form; Difco Laboratories) and 15 g. L -1

Bacto agar (Difco

Laboratories). These plated roots were incubated for 3 weeks

at room temperature (approximately 23°C).

Morphological observation and identification Fungal isolates were identified on the basis of microscopic

morphology. The pure fungal culture was grown at room temperature on 55-mm diameter Petri dishes containing half-strength cornmeal malt yeast extract agar (1/2 CMMY, 25 g

corn meal (infusion from Difco), 15 g Bacto agar (Difco), 10g

malt extract (Difco), 2g yeast extract (Difco), for 1 L distilled water). To provide good observation conditions, slide cultures

were made. Small pieces, approximately 3 x 3 mm, of Publum

agar [Mead Johnson mixed Publum; Canadian Post Corporation, Ontario, Canada, 25 g; Bacto agar, 5 g; MilliQ water, 250 ml] were sandwiched between two 18 x 18-mmcover glasses (Matsunami Class Ind., Osaka, Japan) and

placed in a 9-cm water agar plate to provide humidity. After 2-4

weeks, when the culture had grown sufficiently, the Publum agar was carefully removed and cover glasses were mounted

on 76 x 26 mm micro slide glasses using PVLG (Polyvinyl alcohol, 16.6 g; Lactic acid (Wako Chemical Ind., Osaka,

Japan), 100 ml; glycerin (Wako Chemical Ind., Osaka, Japan), 10 ml; MilliQ water, 100 ml) mounting medium. Conidiogenous

cells and conidia were measured under a light microscope (BX51; Olympus, Tokyo, Japan) with UPlanFLN FLN100x/1.30 Oil.

DNA extraction, amplification, sequencing and analysis Fungal DNA were extracted using a PrepMan TM Ultra

Extraction Kit (Applied Biosystem, Foster City, CA, USA) according to the manufacturer’s protocol, The fungal isolate

was identified by sequencing the internal transcribed region

(ITS) of the 18S rDNA, using universal primers ITS-1 (5'-TCC

GTA GGT GAA CCT GCG G-3') and ITS-4 (5'-TCC TCC GCT TAT TGA TAT GC-3'). Fifty microliters of PCR mixture contained 0.2 μM concentration of each primer, 0.2 mM of eachdeoxynucleoside triphosphate, 10× Ex Taq buffer (TaKaRa Bio,

Otsu, Japan) and 0.25 U of Ex Taq DNA polymerase (TaKaRa

Bio), and 50ng DNA template. The reaction cycle consisted of

initial denaturation at 94 °C for 4 min followed by 35 cycles of

denaturation at 94 °C for 35 sec, annealing at 52 °C for 55 sec and extension at 72 °C for 2 min, and final extension at 72 °Cfor 10 min. The PCR products were sequenced using a model 3130x DNA sequencer (Applied Biosystems, Foster City, CA,USA) and the ABI PRISM TM Big Dye Terminator v3.1 Cycle

Sequencing Ready Reaction Kit (Applied Biosystems). The primers used for sequence determination were ITS1F and ITS4R. The determined sequences were analyzed usingMEGA version 5.05 and compared with similar DNA sequencesretrieved from the DDBJ/EMBL/GenBank databases using the NCBIBLAST program.

Pathogenicity screening In order to distinguish non-pathogenic fungi from pathogenic

and other saprotrophic fungi, 15 fungal isolates showing

diverse morphology were grown on oat meal agar medium (OMA; 10 g oatmeal and 18 g Bacto agar) enriched withnutrients (1 g MgSO 4·7H

2O, 1.5 g KH

2PO

4, and 1 g NaNO

3) per

liter in Petri dishes (55-mm diameter). They were incubated at

room temperature (approximately 23°C). After two weeks, 2-

day-old Chinese cabbage seedlings (three seedlings per plate)were transplanted onto each fungal colony. The seedlings

transplanted onto non-inoculated medium were used as a control. The whole set was placed into sterile culture pots

(CB-1; As One, Osaka, Japan) and incubated in a growth chamber at 23°C under a 16-h photoperiod (180 mol m -2

s -1

) for

2 weeks. Symptoms were evaluated according to an index of 0 to 3 (0: no visible symptoms; 1: light yellowing; 2: yellowing and

late growth; 3: wilting or death). Plants were harvested and oven-dried at 60°C for 48 h and their dry weight was measuredfor comparison with control plants. Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org2 November 2013 | Volume 8 | Issue 11 | e78746 Endophyte screeningAfter successful pathogenicity testing, the efficacy of

selected isolates to promote tomato growth was observed. The

fungal isolates were grown on Petri dishes filled with oat meal

agar medium supplemented with nutrients (MgSO 4.7H

2O, 1 g,

KH 2PO

4 1.5 g; and NaNO

3 1 g L -1

). Surface sterilization of

tomato seeds was performed as described for the

pathogenicity testing. Inoculation was preformed as described above.

Endophyte screening for organic nitrogen sources In order to identify the effect of nitrogen on fungal infection, a

nitrogen source test was conducted. The selected fungal isolates were grown for 2 weeks in 6-cm Petri dishes filled with

oat meal agar medium as described above but NaNO 3, 1g L -1

was replaced by one of the selected nitrogen sources of amino acids, such as L-Valin, L-Phenylalanine, and L-Leucine (Wako Industries, Ltd., Japan), at the concentration of 20 mg L -1

Tomato seedlings (three seedlings per plate) were transplanted and inoculation was preformed as described previously. To determine the endophytic nature of the fungal isolate,

infected hyphae of the inoculated fungi in 3-week-old tomato

seedling roots were observed after washing, cross sectioned,

and stained in 50% acetic acid solution containing 0.005% cotton blue under an Olympus BX50 microscope with

UplanFI20 and 40/0.30 objectives (Olympus).

Data analysis The mean dry biomass of each treatment was calculated and

analyzed with one-way ANOVA. Differences among treatment

means were detected with Tukey’s honestly significant difference test (Tukey HSD).

Results and Discussion Fungal isolation Two hundred and five fungal isolates were obtained from 270

root segments of tomato and Chinese cabbage grown as bait plants in a mixed soil. Fifty-six percent of fungal isolates were

isolated from Chinese cabbage, and 44 % were isolated from

tomato. The dominant isolated fungi are species of Fusarium

(22%). They were mostly isolated within 4 days of placing root

segments on the medium. Other taxa, including Scolecobasidium humicola (4%), Leptodontidium orchidicola

(1%), and Phialocephala fortinii (1%) were isolated in much

smaller numbers from both plants. They were mostly isolated

after one to three weeks. Scolecobasidium humicola were

found at all three sites but L. orchidicola and P. fortinii only in

the forest. Phialocephala fortinii and L.orchidicola are DSE

fungi distributed in a wide geographical area in many alpine and subalpine habitats. In addition, P. fortinii is considered to

be the dominant root endophyte in forests [ 19]. The genus of

Scolecobasidium was first described by Abbott in 1927 as two

species, S. terreum and S. constrictum . The genus includes

soil-borne and saprotrophytic species from plant litter [ 20–23].

Our study indicates that S. humicola is a dark septate

endophyte in forests and agriculture fields, and is able toimprove plant growth in comparison with the control. A new endophytic species, S. humicola, was described for the first

time in this paper.

Endophyte screening To eliminate saprotrophic and/or pathogenic fungal isolates,

Chinese cabbage seedlings were inoculated with 15 randomly selected isolates showing diverse morphology from each colonial morphology group. Seven isolates were originally

obtained from the forest, 5 from a conventional field and 3 from an organic field. In the results of the inoculation test, only two of the 15 isolates tested (approximately 13 %) were notpathogenic to the Chinese cabbage seedlings. These two

isolates, H2-2 and F1-3, caused no visible sign of disease or decay of the seedlings. The weight of dried plants was 56 ± 11

and 59 ± 14 mg for H2-2 and F1-3, respectively, and showed

no significant difference compared to the control plants (68 ± 5 mg) (Figure 1). The most ineffective isolates (over 86%), once

re-inoculated in to axenically-grown Chinese cabbage seedlings, caused extremely yellowing of leaves and

suppression of plant growth (Figure 1). Colonies of 3 isolates, H2-2, F1-3 and O-MH, on OMA

medium were similarly dark brown. The colonial growth of the 3 isolates was similarly slow (up to 15 mm in diameter after 4-

week incubation at 23 °C) (Figure 2a). The conidiophores were

micronematous, and the conidia rough-walled with one to three septata (Figure 2b). Mecelial hyphae were hyaline to brown,

septate-forming densely coiled hyphae, resembling microsclerotia. These morphological characteristics were

identical among the 3 isolates and were in agreement with the

genus of Scolecobasidium .

The ITS sequence of the three isolates showed 99%-100%

similarity to Scolecobasidium humicola (NCBI/GenBank

Accession No. DQ307332.1).

Nitrogen source utilization and anatomic observations Uninoculated (control) plants could use NaNO 3, but could not

effectively use three amino acids (valin, leucine, and phenylalanine). Alternatively, plants treated with three isolates,

H2-2, F1-3, and O-MH, of S. humicola were able to use all 3

amino acids (Figure 3). The treated plants with H2-2 and F1-3

isolates were able to grow well on the medium amended with NaNO 3 as N source, but the treated plants with O-MH isolate

showed yellowing leaves and decreased biomass by 50% on the same medium. The dry weight of treated plants with 3

isolates in Valin treatment was significantly high compared to

the control. When phenylalanine was used with isolate F1-3,

tomato plant biomass was significantly improved but not with 2 isolates H2-2 and O-MH. Optimum plant growth was observed

when the plants were treated with H2-2 and F1-3 amended with leucine as a nitrogen source at 2% concentration. Many studies have suggested the involvement of DSE fungi

in nutrient uptake to host plants and improved plant growth

under natural conditions, i.e. a forest [ 12,13,19]. In addition,

DSE fungi have been isolated from agriculture fields under

nutrient-stressed conditions [ 24,25]. We successfully showed

that tomato plants treated with S. humicola H2-2 and F1-3 were

able to use amino acids and the plant biomass increased in Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org3 November 2013 | Volume 8 | Issue 11 | e78746 comparison with the control. This finding suggested that the

ability of S. humicola as a plant growth-promoting fungus

increased under organic nitrogen sources. This is not a unique

feature of S. humicola as Heteroconium chaetospira could

significantly improve plant biomass by transporting organic nitrogen to host plant [ 26]. Newsham [ 27] confirmed that the

inoculated host plant with the selected DSE fungi responds more positively if there is no supply of available inorganic

nitrogen. To determine the endophytic nature of S. humicola isolate

H2-2, anatomical observation of tomato roots under NaNO 3

leucine treatment was conducted. The hyphae of S. humicola colonized in epidermal cells heavily, but fungal hyphea were

lightly colonized in outer cortical cells. No hyphae could be observed in the inner cortical cells or in the vascular cylinder

under both treatments ( Figure 4). The fungal hyphae only

produced conidia and microsclerotia-like-structures on the root surface under NaNO 3 treatment.

In conclusion, although future detailed research is necessary

to fully support our hypothesis, our study found for the first time that S. humicola is a common DSE species and may act as a

key DSE species under both natural and agricultural conditions.

Figure 1. Dry weight of Chinese cabbage inoculated with different fungal isolates. Dry weight of Chinese cabbage seedlings

grown on basal media (10 g oatmeal and 18 g Bacto agar, enriched with nutrients 1 g MgSO 4·7H

2O, 1.5 g KH

2PO

4, and 1 g NaNO

inoculated with different fungal isolates. The filled columns represent the groups of selected fungi. Data are the means ± SE, n = 5. Asterisks represent significant differences between each treatment and the control (** P <0.01, * P <0.05) following Tukey’s honestlysignificant difference test.

doi: 10.1371/journal.pone.0078746.g001

Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org4 November 2013 | Volume 8 | Issue 11 | e78746 Figure 2. Scolecobasidium humicola colony and

conidia. Colony (A) and a light micrograph (B) of

Scolecobasidium humicola after 14 days at 23°C grown on

OMA medium. Arrowhead indicates a micronematous conidiophore with rough-walled septate conidia (arrows). Bars:

A = 14 mm, B = 20 µm. (C) Tomato seedlings grown on basal

media (OMA) amended with L-Leucine amino acid, the control

on the right , and inoculated tomato seedlings with S.humicola

on the left. doi: 10.1371/journal.pone.0078746.g002

Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org5 November 2013 | Volume 8 | Issue 11 | e78746 Figure 3. Dry weights of tomato inoculated with Scolecobasidium humicola . Dry weights of tomato seedlings grown on basal

media (OMA) amended with four different nitrogen sources. white bar: control, black bar: Scolecobasidium humicola H2-2, black

lines bar: S. humicola F1-3, and gray lines bar: S. humicola O-MH treatments. Data are the means ± SE, n = 5. Asterisks represent

significant differences between each treatment and the control (P <0.05) following Tukey’s honestly significant difference test.

doi: 10.1371/journal.pone.0078746.g003

Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org6 November 2013 | Volume 8 | Issue 11 | e78746 Figure 4. Interaction between tomato roots and Scolecobasidium humicola. Cross section of a tomato root stained with

0.005% cotton blue in 50% acetic acid 3 weeks after inoculation. The cortex (Co) mostly consists of three cell layers. Fungal hyphae

can be seen on the root surface (arrows), within epidermal (Ep) cells. Vc = vascular cylinder. Bar = 20 µm.

doi: 10.1371/journal.pone.0078746.g004

Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org7 November 2013 | Volume 8 | Issue 11 | e78746 Author ContributionsConceived and designed the experiments: RSM KN. Performedthe experiments: RSM. Analyzed the data: RSM. Contributedreagents/materials/analysis tools: RSM KN. Wrote the manuscript: RSM KN. RSM KN.

References

1.Willer H, Kilcher L (2012) The World of Organic Agriculture - Statisticsand Emerging. Trends 2012. Research Institute of Organic Agriculture

(FiBL), Frick, and International Federation of Organic Agriculture

Movements (IFOAM).

Rodrigues MA, Pereira A, Cabanas JE, Dias L, Pires J et al. (2006) Crops use-efficiency of nitrogen from manures permitted in organicfarming. Eur J Agron. 25: 328–335. doi:10.1016/j.eja.2006.07.002.

Reeve JR, Smith JL, Boggs LC, Reganold JP (2008) Soil-based cycling

and differential uptake of amino acids by three species of strawberry (Fragaria spp.) plants. Soil Biol Biochem 40: 2547–2552. doi: 10.1016/

j.soilbio.2008.06.015.

Inselsbacher E, Näsholm T (2012) The below-ground perspective of

forest plants: soil provides mainly organic nitrogen for plants and

mycorrhizal fungi. New Phytol 195(2): 329-334. doi: 10.1111/j.

1469-8137.2012.04169.x. PubMed: 22564239.

Finlay RD, Sodestrom B (1992) Mycorrhiza and carbon flow to the soil. M Allen. Mycorrhiza functioning. London, UK: Chapman & Hall. pp.

134-160.

Hogberg P (1989) Root symbioses of trees in savannas. In: J Proctor. Mineral nutrients in tropical forest and savanna ecosystems. Specialpublication of the British Ecological Society, 9. Oxford, UK: Blackwell

Scientific Publishing House. pp.121-136.

Gosling P, Hodge A, Goodlass G, Bending GD (2006) Arbuscular

mycorrhizal fungi and organic farming. Agriculture Ecosyst Enivronment

113: 17-35. doi:10.1016/j.agee.2005.09.009.

Jumpponen A (2001) Dark septate endophytes are they mycorrhizal?

Mycorrhiza.11: 207–211. doi:10.1007/s005720100112.

Wilson BJ, Addy HD, Tsuneda A, Hambleton S, Currah RS (2004) Phialocephala sphaeroides sp. nov., a new species among the dark

septate endophytes from a boreal wetland in Canada. Can J Bot, 82:

607-617. doi:10.1139/b04-030.

10.

Silvani VA, Fracchia S, Fernández L, Pérgola M, Godeas A (2008) A

simple method to obtain endophytic microorganisms from fieldcollectedroots. Soil Biol Biochem, 40: 1259-1263. doi: 10.1016/j.soilbio.

2007.11.022.

11.

Jumpponen A, Trappe JM (1998) Dark septate endophytes: a review of

facultative biotrophic root-colonizing fungi. New Phytol 140: 295-310. doi:10.1046/j.1469-8137.1998.00265.x.

12.

Haselwandter K, Read DJ (1982) The significance of root-fungus

association in two Carex species of high-alpine plant communities.

Oecologia 53: 352–354. doi:10.1007/BF00389012.

13.

Jumpponen A, Mattson K, Trappe JM (1998) Mycorrhizal functioning of

Phialocephala fortinii with Pinus contorta on glacier fore front soil:interactions with soil nitrogen and organic matter. Mycorrhiza 7: 261–

265. doi:10.1007/s005720050190.

14.

Finlay RD, Frostegård Å, Sonnerfeldt AM (1992) Utilization of organic and inorganic nitrogen sources by ectomycorrhizal fungi in pure culture

and in symbiosis with Pinus contorta Dougl. ex Loud. New Phytol 120:

105–115. doi:10.1111/j.1469-8137.1992.tb01063.x.

15.

Caldwell BA, Jumpponen A (2003) Arylsufatase production by

mycorrhizal fungi. In: Fourth International Conference on Mycorrhizae, Montreal, Canada 312.

16.

Narisawa K, Tokumasu S, Hashiba T (1998) Suppression of clubroot

formation in Chinese cabbage by root endophytic fungus, Heteroconium Chaetospira . Plant Pathol 47: 206-210. doi: 10.1046/j.

1365-3059.1998.00225.x.

17.

Narisawa K, Kawamata H, Currah RS, Hashiba T (2002) Suppression

of Verticillium wilt in eggplant by some fungal root endophytes.Eur J

Plant Pathol 108: 103-109. doi:10.1023/A:1015080311041.

18.

Narisawa K, Usuki F, Hashiba T (2004) Control of Verticillium yellows in

Chinese cabbage by the dark septate endophytic fungus LtVB3. Phytopathology 94: 412-418. doi: 10.1094/PHYTO.2004.94.5.412.

PubMed: 18943758.

19.

Addy HD, Hambleton S, Currah RS (2000) Distribution and molecular

characterization of the root endophyte Phialocephala fortinii along an

environmental gradient in the boreal forest of Alberta. Mycol Res104.

20.

Abott EV (1927) Scolecobasidium, a new genus of soil fungi.

Mycologia: 29-31.

21.

Barron GL, Busch LV (1962) Studies on the soil hyphomycete

Scolecobasidium . Can J Bot. 40: 77–84. doi:10.1139/b62-009.

22.

Roy RY, Dwivedi RS, Mishra RR (1962) Two new species of

Scolecobasidium from soil. Lloydia. 25: 164–166.

23.

Grandi RAP, Gusmão LFP (2002) Decomposing hyphomycetes on leaf

litter of Tibouchina pulchra . Cogn. Rev Bras Bot. 25: 79–87.

24.

Priyadharsini P, Pandey RR, Muthukumar T (2012) Arbuscular

mycorrhizal and dark septate fungal associations in shallot ( Allium cepa

L. var. aggregatum ) under conventional agriculture. Acta Bot Croat 71

(1): 159–175.

25.

Zubek S, Stefanowicz AM, Błaszkowski J (2012) Arbuscular

mycorrhizal fungi and soil microbial communities under contrasting

fertilization of three medicinal plants. Appl Soil Ecol 59: 106–115. doi: 10.1016/j.apsoil.2012.04.008.

26.

Usuki F, Narisawa K (2007) A mutualistic symbiosis between a dark

septate endophytic fungus, Heteroconium Chaetospira, and a

nonmycorrhizal plant, Chinese cabbage. Mycologia, 99(2): 175–184. doi:10.3852/mycologia.99.2.175. PubMed: 17682770.

27.

Newsham KK (2011) A meta-analysis of plant responses to dark

septate root endophytes. New Phytol. doi: 10.1111/j.

1469-8137.2010.03611.x. Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org8 November 2013 | Volume 8 | Issue 11 | e78746