Calibrachoa (Calibrachoa x hybrida) is a highly valued solanaceous flowering ornamental plant, characterized by its drought-hardiness, abundant flowering, and diverse flower colors. Recently, the saprobic soil fungus Mortierella elongata was isolated as a root endophyte from eastern cottonwood (Populus deltoides) and identified as a potential biological amendment for bioenergy and food crops. Two greenhouse experiments were conducted by transplanting rooted cuttings of Calibrachoa cv. ‘Kabloom Deep Blue' into a potting media mixture amended with 1 or 2% volume mix ratio of millet seed colonized with one of four isolates of M. elongata. Plants were assessed weekly for flower production and 86 days post inoculation for leaf/stem, root, and total dry weight. M. elongata strain 624- significantly increased flower production compared to the non-inoculated millet seed controls at 6 and 7 wk post amendment in both experiments. Above and below ground vegetative dry weight for plants grown in potting media mixture amended with M. elongata isolates varied across isolates, fungal amendment concentrations, and experiments. Our results suggest that potting media mixture amended with M. elongata strain 624- can increase flower production of Calibrachoa during peak marketable periods.

Index words: Calibrachoa, plant growth promotion, plant reproduction, biological amendment.

Species used in this study: Calibrachoa (Calibrachoa x hybrida); Mortierella elongata Linnem.

Growers prize Calibrachoa for their abundant production of aesthetically pleasing and colorful flowers, an aspect known to be influenced by fungal endophytes in other plants. Our research suggests that the soil fungus Mortierella elongata can increase the number of flowers produced by Calibrachoa during peak marketable periods. Leaf/stem and root growth varied in response to amendment of the fungus to potting media. The addition of a biological-based amendment that conveys a beneficial and desirable horticultural characteristic has the potential to become a valuable component for increasing marketability of Calibrachoa while also potentially decreasing synthetic fertilizer costs for growers.

The flowering ornamental plant Calibrachoa (Calibrachoa x hybrida) is commonly known as million bells. The genus Calibrachoa Llave and Lex. is an annual or perennial shrub-like groundcover plant with woody stems, exhibiting numerous small trumpet-shaped flowers. Calibrachoa was commercialized due to its desirable horticultural characteristics, including attractive flowers, range of flower colors, drought tolerance, and plant habit. Calibrachoa belongs to the Solanaceae family, is a close relative of petunia and likely originated in southern South America (Olmstead et al. 2008). As of 2014, annual sales of Calibrachoa in the United States were valued at 45 million US dollars (USDA 2014). Calibrachoa are primarily produced as vegetative cuttings in the US with production located in South America and Africa. Growers prize Calibrachoa for its abundant and prolonged flower production. Fungal endophytes have recently been implicated in shortening time to flowering and promoting proliferation of flowers (Das et al. 2012, Ghanem et al. 2014). Fungal endophytes of plants are defined as asymptomatic fungal infections that reside within host tissue for at least a portion of their life cycle without causing apparent harm and commonly occur in most species of plants (Petrini 1991, Strobel & Daisy 2003, Carroll 1988). Beneficial fungal endophytes can increase plant biomass and aid in nutrient acquisition (Khalmuratova et al. 2015, Usuki & Narisawa 2017, Newsham 2011). Endophytic fungi of roots are emerging as a potential alternative to chemical-based amendments and inputs, due to their promotion of plant growth and reduced environmental impact (Kauppinen et al. 2016).

The fungus Mortierella elongata Linnem. is commonly found in soil throughout temperate regions of the world and typically obtains nutrients for growth and development from non-living and decaying organic matter as a saprobe (Wagner et al. 2013, Tedersoo et al. 2014). More recently, M. elongata has been isolated as an endophyte from actively growing roots of field mustard (Brassica campestris L.), common reed [Phragmites australis (Cav.) Trin. ex Seud.] and eastern cottonwood (Populus deltoides W. Bartram ex Marshall) (Khalmuratova et al. 2015, Narisawa et al. 1998, Bonito et al. 2016). Liao et al. (2019) observed an increase in total plant dry weight of black cottonwood (Populus trichocarpa Torr. & Gray) inoculated with M. elongata isolate 93. Mortierella elongata is also known to host bacterial endosymbionts, including Mycoavidus cysteinexigens Ohshima, that have been hypothesized to reduce fungal radial growth rates (Ohshima et al. 2016, Uehling et al. 2017). Other species within the genus Mortierella have been investigated for plant growth promotion properties. For example, M. alpina Peyronel is linked to increased dry weight and number of apical buds in Crocus sativus L. (Wani et al. 2017). Additionally, M. hyalina (Harz) W. Gams was shown to increase aboveground dry weight of Arabidopsis thaliana (L.) Heynh. seedlings (Johnson et al. 2019).

The benefit of a root fungal endophyte such as M. elongata for increasing flowering and plant weight in ornamental plants remains understudied. In this study, we investigated the interaction between the root endophytic fungus M. elongata and Calibrachoa. The primary objective of this study was to determine the effect of M. elongata isolates and fungal amendment concentration on above and below ground plant weight, and flower production of Calibrachoa. We hypothesized that M. elongata will increase plant weight and flowering when amended to potting media mixtures.

Fungal isolates

Two isolates of M. elongata (93+ and 624+) sampled from P. deltoides roots along the Yadkin River in North Carolina by Rytas Vilgalys (Duke University, Durham NC) were used in this study (Bonito et al. 2014). These isolates were derived from internal root tissue and confirmed to harbor endosymbiotic bacteria (Bonito et al. 2016, Uehling et al. 2017). Two additional strains (93- and 624-) generated by treating M. elongata isolates 93+ and 624+ with antibiotics were also used in this study (Uehling et al., 2017). Pure cultures of each isolate and strain of M. elongata were grown on malt extract agar (Becton, Dickinson and Company, Sparks, MD) for approximately 2 wk at 24 C (75.2 F) under photoperiod with a 12 h light/dark cycle (48 μE m−2 s−1). Isolates and strains of M. elongata were maintained in long-term storage in sterilized distilled water at 23 C (73.4 F) and -80 C (-112 F) in a sterile 25% glycerol solution.

Production of M. elongata for greenhouse experiments

Millet seed [300 g (10.6 oz)] was mixed with an equal volume of distilled H2O [300 ml (10.1 oz)] in a 3.79 L (1 gal) plastic jug, plugged with a foam stopper capped with aluminum foil and autoclaved for 45 min at 121 C (249.8 F) (124 kPa) for three consecutive days. After plastic jugs with millet seed reached room temperature, half of a 9-cm (3.5 in) diameter petri dish of each isolate or strain grown on malt extract agar was cut into small plugs and aseptically added to each jug. Plastic jugs were incubated at room temperature 21 C ± 2 C (69.8 F ± 4 F) under fluorescent light for 4 wk. Millet seed amended with one half of a 9-cm (3.5 in) diameter petri dish of malt extract agar without fungus served as a control.

Greenhouse experiments

Tip cuttings 4-6 cm (1.6-2.4 in) in length and less than one week old of Calibrachoa cv. ‘Kabloom Deep Blue' were obtained from Ball Seed Co. (Chicago IL). Tip cuttings were dipped in a rooting hormone powder (Root Boost with 0.1% active ingredient indole-3 butyric acid, Tech Pac, LLC. Lexington KY). Cuttings were placed in soilless peat-based potting media (Farfard 2P, Sun Gro Horticulture, Agawam, MA) in 128 cell flats for 3 wk. Plants were misted intermittently to promote root formation in a greenhouse at North Carolina State University, Raleigh. Rooted cuttings of Calibrachoa were transplanted individually into 10-cm (4 in) square pots, with a 2:1:1 ratio of Farfard 2P potting media, pasteurized soil (50 C/122 F for 30 min), and pasteurized sand. Twenty-four hours prior to transplanting, the potting media mixture was amended with millet seed colonized by individual isolates (93+ or 624+), or strains (624- or 93-) of M. elongata at a 1% or 2% volume mix ratio (v/v). The millet seed and potting media mix was thoroughly homogenized by agitation. Controls consisted of non-inoculated millet seed added at 1 or 2% v/v and a non-amended control. All plants were fertilized with one teaspoon of Osmocote (14N–6.2P–11.6K) to provide minimal fertilization. There were three replicates of five plants per each treatment arranged in a randomized complete block design. The experiment was conducted twice. Experiment 1 was initiated on March 18 and harvested June 11, 2017. Experiment 2 was initiated on July 9 and harvested October 2, 2017. Minimum and maximum greenhouse temperatures were recorded daily throughout the duration of the experiments.

Plant growth assessment

Since flowering is an important horticultural production trait for Calibrachoa, the number of fully emerged flowers per plant was counted weekly following flower emergence from wk 2 to 11. Twelve weeks after transplanting, leaves, stems and flowers were harvested by clipping each plant stem at the surface of the potting media mixture and placing plant tissue into a paper bag. Roots from each plant were gently rinsed under running tap water to remove excess potting media mixture. Leaves/stems (foliage) and roots were dried separately in paper bags for 8 h at 50 to 60 C (122 to 140 F) in a Thelco drying oven (Precision Scientific Company, Chicago IL). Dried foliage and roots were weighed separately using a Metler Toledo ME4002E scale (Columbus, OH). Plant tissue analysis of dried leaves from individual plants collected from experiments post-harvest were analyzed at the North Carolina Department of Agriculture (NCDA, Raleigh NC). 2 plants were selected from each treatment group for individual analysis.

Statistical analysis

All statistical analyses were performed using SAS (version 9.4, SAS Institute, Cary NC). Mean plant dry weight, flower metrics, and plant tissue analysis data from experiment 1 and 2 were subject to analysis of variance (ANOVA) using the PROC GLM procedure of SAS. A generalized linear model was utilized for continuous and count data for leaves/stems, root weight, and flowering assuming a normal distribution for continuous data and Poisson distribution for count data (Stroup, 2015). When ANOVA was significant for the isolate by amendment volume interaction, simple effects were examined to compare isolates by individual amendment volumes. Post hoc analysis of data was performed with Tukey's honestly significant difference test to examine significant differences among means (alpha= 0.05).

Greenhouse experiment 1

Leaf/stem, root, and total dry weight differed significantly by isolate and there was a significant amendment volume by isolate interaction for each plant growth parameter assessed (P<0.002). Therefore, treatments were analyzed for each amendment volume separately to examine simple effects. This resulted in comparison of fungal treatments to non-inoculated controls of sterile millet seed of the same amendment volume. Rooted cuttings grown in the potting media mixture amended with M. elongata strain 624- exhibited higher leaf/stem (P<0.0001), root (P=0.0005), and total (P<0.0001) dry weight compared to the non-inoculated millet seed control at 1% v/v (Fig. 1). In contrast, cuttings grown in potting media mixture amended with M. elongata isolate 624+ exhibited significantly lower dry weight (P< 0.0001) for all three dependent variables (leaf/stem, root and total dry weight) at 1% v/v. Rooted cuttings amended with M. elongata isolate 93+ at a 2% inoculum volume exhibited significantly lower leaf/stem, root, and total dry weight compared to the non-amended 2% v/v controls (P< 0.0001, = 0.0096, and =0.0001, respectively) (Fig. 2). Significant differences among isolates were observed for flower production for wk 5, 6 and 7. The isolate by amendment volume interaction was significant (P<0.018) for flower production in wk 5, 6 and 7, hence experiments were analyzed separately for each amendment volume to determine simple effects. Rooted cuttings grown in potting media mixture amended with M. elongata strain 624- at 1% v/v produced significantly more flowers than the 1% v/v non-inoculated millet seed control at 5 (P<0.0001), 6 (P=0.0007) and 7 (P< 0.0001) wk (Fig. 3). Rooted cuttings planted in potting media mixture amended with M. elongata isolate 624+ and 93+ at 1% v/v produced significantly less flowers than the 1% v/v non-inoculated millet seed control at weeks 6 and 7 (P= <0.04) (Fig. 3). Calibrachoa cuttings produced in potting media mixture amended with M. elongata strain 624- at 2% v/v produced significantly more flowers than the 2% v/v non-inoculated millet seed control at weeks 5 and 6 post inoculation (P= 0.0001) (Fig. 3).

Fig. 1

Root, foliage (leaf/stem), and total dry weight of Calibrachoa cultivar ‘Kabloom Deep Blue' grown in potting media mixture amended with a single isolate (624+ or 93+) or strain (624- or 93-) of M. elongata at 1% millet seed amendment volume for experiment 1. Plants were harvested at 12 weeks post inoculation. Dried foliage and roots were weighed separately. C1 represents the non-inoculated millet seed control at 1% v/v.

Fig. 1

Root, foliage (leaf/stem), and total dry weight of Calibrachoa cultivar ‘Kabloom Deep Blue' grown in potting media mixture amended with a single isolate (624+ or 93+) or strain (624- or 93-) of M. elongata at 1% millet seed amendment volume for experiment 1. Plants were harvested at 12 weeks post inoculation. Dried foliage and roots were weighed separately. C1 represents the non-inoculated millet seed control at 1% v/v.

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Fig. 2

Root, foliage (leaf/stem), and total dry weight of Calibrachoa cultivar ‘Kabloom Deep Blue' grown in potting media mixture amended with a single isolate (624+ or 93+) or strain (624- or 93-) of M. elongata at 2% millet seed amendment volume for experiment 1. Plants were harvested at 12 weeks post inoculation. Dried foliage and roots were weighed separately. C1 represents the non-inoculated millet seed control at 2% v/v.

Fig. 2

Root, foliage (leaf/stem), and total dry weight of Calibrachoa cultivar ‘Kabloom Deep Blue' grown in potting media mixture amended with a single isolate (624+ or 93+) or strain (624- or 93-) of M. elongata at 2% millet seed amendment volume for experiment 1. Plants were harvested at 12 weeks post inoculation. Dried foliage and roots were weighed separately. C1 represents the non-inoculated millet seed control at 2% v/v.

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Fig. 3

Means of flower production of Calibrachoa cultivar ‘Kabloom Deep Blue' amended with individual isolate (624+ or 93+) or strain (624- or 93-) of M. elongata on millet seed and non-inoculated millet seed controls. Data presented for each treatment is an average of 1% and 2% amendment volumes. Flower production assessed weekly at 2 wk to 11 wk post inoculation in experiment 1.

Fig. 3

Means of flower production of Calibrachoa cultivar ‘Kabloom Deep Blue' amended with individual isolate (624+ or 93+) or strain (624- or 93-) of M. elongata on millet seed and non-inoculated millet seed controls. Data presented for each treatment is an average of 1% and 2% amendment volumes. Flower production assessed weekly at 2 wk to 11 wk post inoculation in experiment 1.

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Greenhouse experiment 2

Leaf/stem, root, and total dry weight differed significantly among isolates. There was a significant isolate by amendment volume interaction for each metric (P< 0.05). Therefore, isolates were analyzed by amendment volume to examine simple effects. Rooted cuttings amended with M. elongata strains 93+, 624+, and 624- exhibited slightly lower leaf/stem, root, and total dry weight compared to the non-inoculated millet seed control at 1% v/v. Cuttings grown in potting media mixture amended with M. elongata strain 93- at 1% v/v had significantly lower leaf/stem (P=0.0103) and total (P=0.0143) dry weight compared to the non-inoculated millet seed control at 1% v/v (Fig. 4). At the 2% inoculum volume, rooted cuttings grown in potting media mixture amended with M. elongata isolates did not exhibit significant differences in leaf/stem, root and total dry weight compared to the non-inoculated millet seed control at 2% v/v. Non-significant trends indicated that 93- and 624- exhibited slightly greater foliage, root, and total dry weight compared to the non-inoculated millet seed control, while 93+ and 624+ exhibited slightly lower foliage, root, and total dry weight compared to the non-inoculated millet seed control (Fig. 5). Flower production for all fungal inoculum amendment volumes and isolates varied across weeks 2 through 11 of the experiment (Fig. 6). A significant (P< 0.025) isolate by amendment volume interaction for flower production was observed during weeks 4, 6 and 7 and isolates were examined separately for each amendment volume to determine simple effects. Rooted cuttings grown in potting media mixture amended with M. elongata strain 624- at 2% v/v produced significantly more flowers than the non-inoculated millet seed control at weeks 6 and 7 (P< 0.0001) (Fig. 6).

Fig. 4

Root, foliage (leaf/stem), and total dry weight of Calibrachoa cultivar ‘Kabloom Deep Blue' grown in potting media mixture amended with a single isolate (624+ or 93+) or strain (624- or 93-) or M. elongata at 1% millet seed amendment volume for experiment 2. Plants were harvested at 12 weeks post inoculation. Dried foliage and roots were weighed separately. C1 represents the non-fungus inoculated millet seed control at 1% v/v.

Fig. 4

Root, foliage (leaf/stem), and total dry weight of Calibrachoa cultivar ‘Kabloom Deep Blue' grown in potting media mixture amended with a single isolate (624+ or 93+) or strain (624- or 93-) or M. elongata at 1% millet seed amendment volume for experiment 2. Plants were harvested at 12 weeks post inoculation. Dried foliage and roots were weighed separately. C1 represents the non-fungus inoculated millet seed control at 1% v/v.

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Fig. 5

Root, foliage (leaf/stem), and total dry weight of Calibrachoa cultivar ‘Kabloom Deep Blue' grown in potting media mixture amended with single isolate (624+ or 93+) or strain (624- or 93-) of M. elongata at a 2% millet seed amendment volume for experiment 2. Plants were harvested at 12 weeks post inoculation. Dried foliage and roots were weighed separately. C1 represents the non-inoculated millet seed control at 2% v/v.

Fig. 5

Root, foliage (leaf/stem), and total dry weight of Calibrachoa cultivar ‘Kabloom Deep Blue' grown in potting media mixture amended with single isolate (624+ or 93+) or strain (624- or 93-) of M. elongata at a 2% millet seed amendment volume for experiment 2. Plants were harvested at 12 weeks post inoculation. Dried foliage and roots were weighed separately. C1 represents the non-inoculated millet seed control at 2% v/v.

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Fig. 6

Means of flower production of Calibrachoa cv. ‘Kabloom Deep Blue' amended with individual isolate (624+ or 93+) or strain (624- or 93-) of M. elongata on millet seed and non-inoculated millet seed controls. Data presented for each treatment is an average of 1% and 2% amendment volumes. Flower production assessed weekly at 2 wk to 11 wk post inoculation in experiment 2.

Fig. 6

Means of flower production of Calibrachoa cv. ‘Kabloom Deep Blue' amended with individual isolate (624+ or 93+) or strain (624- or 93-) of M. elongata on millet seed and non-inoculated millet seed controls. Data presented for each treatment is an average of 1% and 2% amendment volumes. Flower production assessed weekly at 2 wk to 11 wk post inoculation in experiment 2.

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The application and identification of novel fungal root endophytes with beneficial and desirable plant growth responses represent a growing area of interest for plant scientists (Le Cocq et al. 2016, Lugtenberg et al, 2016). In this study, Calibrachoa plants grown in potting media mixture amended with M. elongata strain 624- had a greater total number of flowers during weeks 6 and 7 in both experiments compared to non-inoculated controls. We assessed flower production on a temporal scale rather than a single time point to more accurately reflect the Calibrachoa production system and timeframe of flowering that corresponds to grower demands for marketable plants. An association between amendment of the fungal endophyte Piriformospora indica Sav. Verma, Aj. Varma, Rexer, G. Kost & P. Franken and increased flower or reproductive structure production has been previously reported in Indian coleus (Das et al. 2012) and cyclamen (Ghanem et al. 2014).

The association of a diverse suite of endosymbiotic bacteria within endophytic fungi has been previously documented (Hoffman & Arnold, 2010). Variation of plant growth response to individual isolates of M. elongata depended largely on presence or absence of endosymbiotic bacteria. Our research suggests that the presence of endosymbiotic bacteria within M. elongata did not enhance plant growth promotion properties of the fungus. Radial hyphal growth of M. elongata isolate AG77 significantly decreases (P<0.05) when associated with the bacterial endosymbionts compared to an antibiotic cured strain of M. elongata AG77, suggesting a metabolic cost of harboring the endosymbiotic bacteria (Uehling et al., 2017). It is not known specifically how the endosymbiotic bacterial community is composed within M. elongata isolates 624+ and 93+, or if the community composition changes over time and/or responds to environmental factors. A direct link between the presence and function of endosymbiotic bacteria sourced from endophytic fungi and plant growth has yet to be firmly established.

Promotion of plant weight by root-associated fungal endophytes is well documented (Rodriguez et al., 2009, Mandyam et al., 2013, Schulz et al., 2002, Ansari et al., 2013, Newsham, 2011). In our study, addition of M. elongata strain 624- increased plant dry weight in experiment 1, but a similar increase in plant dry weight was not observed in experiment 2 for the 1% fungal amendment volume. In general, Calibrachoa in experiment 1 exhibited higher leaf/stem, root, and total plant dry weight compared to plants in experiment 2. In both experiments, significant differences were observed among cuttings planted in potting media mixture amended with 1% and 2% v/v inoculated or non-inoculated millet seed for leaf/stem, root, and total dry weight. Rooted cuttings amended with 2% v/v inoculated or non-inoculated millet seed exhibited significantly higher dry weight for all three metrics compared to rooted cuttings amended with 1% v/v inoculated or non-inoculated millet seed. Cuttings grown in potting media mixture amended with 1% v/v inoculated or non-inoculated millet seed exhibited significantly higher dry weight for all three metrics compared to non-amended controls. Among the controls, inoculum volume 1% and 2% exhibited significantly higher leaf/stem, root, and total dry weight when compared to control volume 0% (P <0.0001 for foliage, root, and total weight).

Plant tissue analysis revealed minor differences among treatments for various nutrients. Significant differences were observed with the element boron, in which adding isolate 93- resulted in significantly greater concentrations compared to the non-amended/non-inoculated controls (P< 0.05). The observed variation in plant growth metrics among M. elongata isolates and experiments in this study suggests that environmental factors and amendment volume may affect root and leaf/stem dry weight. Greenhouse air temperatures differed between the two experiments, with mean minimum and maximum air temperatures for experiment 1 of 20.7 and 34.2 C (69.3 and 93.6 F) and experiment 2 of 22.8 and 36.5 C (73.0 and 97.7 F), respectively. Gams et al. (1972) demonstrated that growth and survival of M. elongata isolates in pure culture are inhibited by temperatures at or above 35 C (95 F). In floriculture plants, higher mean daily air temperatures are inversely related to plant weight (Vaid & Runkle, 2014). The temperatures observed during the second experiment may have decreased fungal growth, although we did not examine or determine optimal fungal growth temperatures in planta. Further research is needed to establish optimal environmental conditions for root colonization by M. elongata.

The floriculture industry relies on consistent production of aesthetically valuable flowering plants. Biological-based amendments are growing in popularity in greenhouse production systems and therefore increasing in market value (Bailey et al. 2010). Fungal endophytes represent novel biological-based sources for increasing plant growth and health with broad applications across agriculture and horticulture. Our studies suggest that M. elongata strain 624- has the potential to increase flowering during marketable periods of Calibrachoa and become a valuable component of biological-based amendments. Future experiments could expand the evaluation of flower promotion effects of M. elongata on other Calibrachoa cultivars and additional plant hosts. Experiments to examine the sensitivity of M. elongata to commonly applied chemical products under floriculture production conditions are also necessary for effective deployment. Our results represent the first report of the fungal endophyte M. elongata increasing flower production in an ornamental plant species.

Ansari,
M.W.,
Trivedi
D.K.,
Sahoo
R.K.,
Gill
S.S.,
and
Tuteja
N.
2013
.
A critical review on fungi mediated plant responses with special emphasis to Piriformospora indica on improved production and protection of crops
.
Plant Physiology and Biochemistry
70
:
403
410
.
Bailey,
K.L.,
Boyetchko
S.M.,
and
Längle
T.
2010
.
Social and economic drivers shaping the future of biological control: A Canadian perspective on the factors affecting the development and use of microbial biopesticides
.
Biological Control
52
:
221
229
.
Bonito,
G.,
Hameed
K.,
Ventura
R.,
Krishnan
J.,
Schadt
C.W.,
and
Vilgalys
R.
2016
.
Isolating a functionally relevant guild of fungi from the root microbiome of Populus
.
Fungal Ecology
22
:
35
42
.
Bonito,
G.,
Reynolds
H.,
Robeson
M.S.,
Nelson
J.,
Hodkinson
B.P.,
Tuskan
G.,
Schadt
C.W.,
and
Vilgalys
R.
2014
.
Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants
.
Molecular Ecology
23
:
3356
3370
.
Carroll,
G.
1988
.
Fungal endophytes in stems and leaves - from latent pathogen to mutualistic symbiont
.
Ecology
69
:
2
9
.
Das,
A.,
Kamal
S.,
Shakil
N.A.,
Sherameti
I.,
Oelmüller
R.,
Dua
M.,
Tuteja
N.,
Johri
A.K.,
and
Varma
A.
2012
.
The root endophyte fungus Piriformospora indica leads to early flowering, higher biomass and altered secondary metabolites of the medicinal plant, Coleus forskohlii
.
Plant Signaling & Behavior
7
:
103
112
.
Gams,
W.,
Chien
C.,
and
Domsch
K.H.
1972
.
Zygospore formation by the heterothallic Mortierella elongata and a related homothallic species, M. epigama sp. nov
.
Transactions of the British Mycological Society
58
:
5
13
.
Ghanem,
G. A.
Ewald,
S. Zerche,
and
Hennig
F.
2014
.
Effect of root colonization with Piriformospora indica and phosphate availability on the growth and reproductive biology of a Cyclamen persicum cultivar
.
Scientia Horticulturae
172
:
233
241
.
Hoffman
M.T.
and
Arnold
A.E.
2010
.
Diverse bacteria inhabit living hyphae of phylogenetically diverse fungal endophytes
.
Applied and Environmental Microbiology
76
:
4063
4675
.
Johnson,
J.M.,
Ludwig
A.,
Furch
A.C.U.,
Mithöfer
A.,
Scholz
S.,
Reichelt
M.,
and
Oelmüller
R.
2019
.
The beneficial root-colonizing fungus Mortierella hyalina promotes the aerial growth of Arabidopsis and activates calcium-dependent responses that restrict Alternaria brassicae-induced disease development in roots
.
Molecular Plant Microbe Interactions doi: 10.1094/MPMI-05-18-0115-R.
Kauppinen,
M.,
Saikkonen
K.,
Helander
M.,
Pirtillä
A.M.,
and
Wäli
P.R.
2016
.
Epichloë grass endophytes in sustainable agriculture
.
Nature Plants
2
:
15224
Khalmuratova,
I.,
Kim
H.,
Nam
Y.J.,
Oh
Y.,
Jeong
M.J.,
Choi
H.R.,
You
Y.H.,
Choo
Y.S.,
Lee
I.J.,
Shin
J.H.,
Yoon
H.,
and
Kim
J.G.
2015
.
Diversity and plant growth promoting capacity of endophytic fungi associated with halophytic plants from the west coast of Korea
.
Mycobiology
43
:
373
383
.
Le Cocq,
K.,
Gurr
S.J.,
Hirsch
P.R.,
and
Mauchline
T.H.
2016
.
Exploitation of endophytes for sustainable agricultural intensification
.
Molecular Plant Pathology
18
:
469
473
.
Liao,
H.L.,
Bonito
G.,
Rojas
J.A.,
Hameed
K.,
Wu
S.,
Schadt
C.W.,
Labbe
J.,
Tuskan
G.A.,
Martin
F.,
Grigoriev
I.V.,
and
Vilgalys
R.
2019
.
Fungal endophytes of Populus trichocarpa alter host phenotype, gene expression and rhizobiome composition
.
Molecular Plant-Microbe Interactions
32
:
853
864
.
Lugtenberg,
B.J.,
Caradus
J.R.,
and
Johnson
L.G.
2016
.
Fungal endophytes for sustainable crop production
.
FEMS Microbiology Ecology
92
:
fiw194.
Mandyam,
K.G.,
Roe
J.,
and
Jumpponen
A.
2013
.
Arabidopsis thaliana model system reveals a continuum of responses to root endophyte colonization
.
Fungal Biology
117
:
250
260
.
Narisawa,
K.,
Tokumasu
S.,
and
Hashiba
T.
1998
.
Suppression of clubroot formation in Chinese cabbage by the root endophytic fungus, Heteroconium chaetospira
.
Plant Pathology
47
:
206
210
.
Newsham
K.K.
2011
.
A meta-analysis of plant responses to dark septate root endophytes
.
New Phytologist
190
:
783
793
.
Ohshima,
S.,
Sato
Y.
,
Fujimura
R.,
Takashima
Y.,
Hamada
M.,
Nishizawa
T.,
Narisawa
K.,
and
Ohta
H.
2016
.
Mycoavidus cysteinexigens gen. nov., sp. nov., an endohyphal bacterium isolated from a soil isolate of the fungus Mortierella elongata
.
International Journal of Systematic Evolutionary Microbiology
66
:
2052
7
.
Olmstead
RG,
Bohs
L.,
Migid
H.A.,
Santiago-Valentin
E.,
Garcia
V.F.
and
Collier
S.M.
2008
.
A molecular phylogeny of the Solanaceae
.
Taxon
57
:
1159
1181
.
Petrini,
O.
1991
.
Fungal endophytes of tree leaves
.
p.
179
197
In:
Andrews,
J.H.
and
Hirano
S.S.
(Eds.)
Microbial ecology of leaves
.
New York
:
Springer
.
Rodriguez,
R.J.,
White
J.F.
Jr
.,
Arnold
A.E.,
and
Redman
R.S.
2009
.
Fungal endophytes: diversity and functional roles
.
New Phytologist
182
:
314
330
.
Schulz,
B.,
Boyle
C.,
Draeger
S.,
Römmert
A.K.,
and
Krohn
K.
2002
.
Endophytic fungi: a source of novel biologically active secondary metabolites
.
Mycological Research
106
:
996
1004
.
Strobel,
G.
and
Daisy
B.
2003
.
Bioprospecting for microbial endophytes and their natural products
.
Microbiology and Molecular Biology Reviews
67
:
491
502
.
Stroup,
W.W.
2015
.
Rethinking the analysis of non-normal data in plant and soil science
.
Agronomy Journal
107
:
811
827
.
Tedersoo,
L.,
Bahram
M.,
Polme
S.,
Köljalg
U.,
Yorou
N.S.,
Wijesundera
R.,
Villarreal
R.L.,
Vasco-Palacios
A.M.,
Thu
P.Q.,
Suija
A.,
Smith
M..E,
Sharp
C.,
Saluveer
E.,
Saitta
A.,
Rosas
M.,
Riit
T.,
Ratkowsky
D.,
Pritsch
K.,
Põldmaa
K.,
Piepenbring
M.,
Phosri
C.,
Peterson
M.,
Parts
K.,
Pärtel
K.,
Otsing
E.,
Nouhra
E.,
Njouonkou
A.L.,
Nilsson
R.H.,
Morgado
L.N.,
Mayor
J.,
May
T.W.,
Majuakim
L.,
Lodge
D.J.,
Lee
S.S.,
Larsson
K.H.,
Kohout
P.,
Hosaka
K.,
Hiiesalu
I.,
Henkel
T.W.,
Harend
H.,
Guo
L.D.,
Greslebin
A.,
Grelet
G.,
Geml
K.J.,
Gates
G.,
Dunstan
W.,
Dunk
C.,
Drenkhan
R.,
Dearnaley
J.,
De Kesel
A.,
Dang
T.,
Chen
X.,
Buegger
F.,
Brearley
F.Q.,
Bonito
G.,
Anslan
S.,
Abell
S.,
and
Abarenkov
K.
2014
.
Fungal biogeography. Global diversity and geography of soil fungi
.
Science
346
:
1256688
.
Uehling,
J.,
Gryganskyi
A.,
Hameed
K.,
Tschaplinski
T.,
Misztal
P.K.,
Wu
S.,
Desiro
A.,
Vande Pol
N.,
Du
Z.,
Zienkiewicz
A.,
Zienkiewicz
K.,
Morin
E.,
Tisserant
E.,
Splivallo
R.,
Hainaut
M.,
Henrissat
B.,
Ohm
R.,
Kuo
A.,
Yan
J.,
Lipzen
A.,
Nolan
M.,
LaButti
K.,
Barry
K.,
Goldstein
A.H.,
Labbé
J.,
Schadt
C.,
Tuskan
G.,
Grigoriev
I.,
Martin
F.,
Vilgalys
R.,
and
Bonito
G.
2017
.
Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens
.
Environmental Microbiology
19
:
2964
2983
.
USDA.
2014
.
Census of Horticultural Specialties 2014
.
p.
6
.
Usuki,
F.
and
Narisawa
K.
2017
.
A mutualistic symbiosis between a dark septate endophytic fungus, Heteroconium chaetospira, and a nonmycorrhizal plant, Chinese cabbage
.
Mycologia
99
:
175
184
.
Vaid
T.M.
and
Runkle
E.S.
2014
.
Mean daily temperature regulates plant quality attributes of annual ornamental plants
.
HortScience
49
:
574
580
.
Wagner,
L.,
Stielow
B.,
Hoffmann
K.,
Petkovits
T.,
Papp
T.,
Vágvölgyi
C.,
de Hoog
G.S.,
Verkley
G.,
and
Voigt
K.
2013
.
A comprehensive molecular phylogeny of the Mortierellales (Mortierellomycotina) based on nuclear ribosomal DNA
.
Persoonia
30
:
77
93
.
Wani,
Z.A.,
Kumar
A.,
Sultan
P.,
Bindu
K.,
Riyaz-Ul-Hassan
S.,
and
Ashraf
N.
2017
.
Mortierella alpina CS10E4, an oleaginous fungal endophyte of Crocus sativus L. enhances apocarotenoid biosynthesis and stress tolerance in the host plant
.
Scientific Reports
7
:
8598
.

Author notes

1

Acknowledgement: This study was supported by Horticultural Research Institute grant # 26597099 awarded to LEB and MAC. The authors would like to thank Dr. Mike Klopmeyer at Ball Horticultural Company for providing plants, Dr. Helen Kraus for providing greenhouse space for rooting cuttings and Dr. Barbara Shew for statistical assistance. The authors also thank Camilo Parada for critical pre-submission review of the manuscript.

2

Department of Entomology and Plant Pathology, Center for Integrated Fungal Research, North Carolina State University. [email protected], corresponding author.