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EVALUATION OF A MYCORRHIZAL-LIKEFUNGUS, PIRIFORMOSPORA INDICA, ONFLORICULTURE CROPSAllison JusticeClemson University, alleybmp13@aol.com

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Recommended CitationJustice, Allison, "EVALUATION OF A MYCORRHIZAL-LIKE FUNGUS, PIRIFORMOSPORA INDICA, ON FLORICULTURECROPS" (2014). All Dissertations. 1313.https://tigerprints.clemson.edu/all_dissertations/1313

EVALUATION OF A MYCORRHIZAL-LIKE FUNGUS, PIRIFORMOSPORA INDICA, ON FLORICULTURE CROPS

A Dissertation Presented to

the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree

Doctor of Philosophy Plant and Environmental Science

by Allison Hope Justice

August 2014

Accepted by: James E. Faust, Committee Chair

Julia L Kerrigan Jeff Adelberg

Vijay Rapaka

ABSTRACT

Piriformospora indica is a fungal endophyte, often called an arbuscular

mycorrhizal-like fungus, that has been shown to provide benefits to plant symbionts by

increasing nutrient uptake, biomass production, flower number, and disease resistance in

a wide range of plant hosts. Research was carried out to investigate the ability of

P. indica to improve plant production in floriculture crops. The first objective was to

determine the optimal environmental conditions for growing P. indica in pure culture.

Environmental conditions were optimized to produce the maximum chlamydospores for

inoculum preparation (Chapter 1). These findings were used in the remaining chapters to

prepare inocula for experiments. The addition of P. indica to unrooted cuttings was

evaluated for enhancement of adventitious root formation (Chapter 2). Benefits of

P. indica inoculation on unrooted cuttings varied between plant species, cultivars and

inoculum concentration. Calibrachoa, impatiens, and petunia plants inoculated with

P. indica were evaluated for growth enhancement and nutrient uptake under nitrogen and

phosphorus restriction, because these are essential nutrients in greenhouse crops (Chapter

3). Our results suggested that inoculation with P. indica can enhance nitrogen uptake,

biomass, and flower number of the three floriculture species tested when nitrogen was

restricted, whereas only calibrachoa demonstrated benefits from inoculation with

P. indica when phosphorus was restricted. Finally, five commercial biological fungicides

and P. indica were evaluated for plant growth promotion and disease suppression of

Phytophthora nicotianae on three petunia species. Petunia × hybrida did not respond to

biological fungicide treatments for growth enhancement or disease suppression. Some

ii

biological fungicide treatments suppressed disease symptoms on Petunia grandiflora and

Petunia multiflora at days 2, 6, 8 and 10 after pathogen inoculation. Improved plant

growth was also dependent on host and biological control agent applied. Our results

showed that inoculation of P. indica on floriculture crops can promote adventitious root

formation on unrooted cuttings, enhance biomass, increase nutrient uptake, and slow

down disease progression of P. nicotianae depending on inoculation levels and plant

species.

iii

DEDICATION

This work is dedicated to Deborah Justice, my mother, who always believes,

loves, and supports without falter.

iv

ACKNOWLEDGMENTS

Though only my name appears on the cover of this dissertation, a great many people have contributed to its production. I owe my gratitude to all those people who have made this dissertation possible and because of whom my graduate experience has been one that I will cherish always. First I would like to thank my family, Deborah, Tom, Mandy, Goob, Matt, Nellie, and my precious nieces for their constant support, encouragement, and love throughout this journey. I simply couldn’t be in the position I am today without having you all in my life. In addition, I would like to thank my animal family, my cat and dog, for being the best roommates, friends, listeners, and waiting every day for me with smiles. I wouldn’t be where I am today without the support and welcomed distraction from my friends whom I consider my second family.

My deepest thanks are to my advisor, Dr. James E. Faust. I have been fortunate to have an advisor who gave me the freedom to explore on my own and at the same time the guidance to recover when my steps faltered. Dr. Faust taught me how to question thoughts and express ideas.

I give special thanks to my committee members, Dr. Julia Kerrigan, Dr. Jeffery Adelberg, and Dr. Vijay Rapaka for their guidance and support not only pertaining to my degree but also their specialties. I will never forget the extra time and kindness given to advance me as a well-rounded horticulturalist. In addition, I would like to thank Dr. Steve Jeffers for his help and guidance.

I would especially like to thank Kelly Lewis, Dan Hunnicutt, and John Wells for taking me under their wing to show a very inexperienced scientist the ropes in a horticulture lab and helping to turn me into a knowledgeable professional. I would also like to thank them for their friendship and constant smiles.

I would like to thank my fellow graduate students and lab mates that were always there to listen and understand the daily problems of a graduate student and provide friendship and continual banter… Jeremy Crook, Travis Higginbotham, and Misty Shealy.

The Fred C. Gloeckner Foundation deserves special thanks for their support financially throughout my time in graduate school.

Last I would like to thank everyone else in the Clemson family. I could not have asked for a more friendly staff and knowledgeable educators. I hope I can one day pass on these tools and life lessons I have learned during my time at Clemson University.

v

TABLE OF CONTENTS

Page

TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii DEDICATION ................................................................................................................ iv ACKNOWLEDGMENTS ............................................................................................... v LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES ......................................................................................................... x CHAPTER I. GROWTH OPTIMIZATION OF AN ARBUSCULAR MYCORRHIZAL-LIKE FUNGUS, PIRIFORMOSPORA INDICA ............................................................ 1 Abstract .................................................................................................... 1 Introduction .............................................................................................. 2 Materials & Methods ............................................................................... 4 Results & Discussion ............................................................................... 7 Conclusion ............................................................................................. 10 Literature Cited ...................................................................................... 14 II. EVALUATING THE POTENTIAL OF A NEW MYCORRHIZAL-LIKE FUNGUS, PIRIFORMOSPORA INDICA, TO IMPOROVE ADVENTITIOUS ROOT FORMATION OF FLORICULTURE CROPS ............................................................. 17 Abstract .................................................................................................. 17 Introduction ............................................................................................ 18 Materials & Methods ............................................................................. 20 Results .................................................................................................... 23 Discussion & Conclusion ....................................................................... 25 Literature Cited ...................................................................................... 31

vi

TABLE OF CONTENTS (CONTINUED) Page

III. RESPONSE OF FLORICULTURE CROPS TO NITROGEN AND PHOSPHORUS RESTRICTION WHEN COLONIZED WITH THE MYCORRHIZAL-LIKE FUNGUS, PIRIFORMOSPORA INDICA ........................................................... 35 Abstract .................................................................................................. 35 Introduction ............................................................................................ 36 Materials and Methods ........................................................................... 40 Results .................................................................................................... 44 Discussion & Conclusion ....................................................................... 46 Literature Cited ...................................................................................... 52 IV. THE POTENTIAL FOR THE FUNGAL ENDOPHYTE PIRIFORMOSPORA INDICA TO PROTECT PETUNIA FROM INFECTION BY PHYTOPHTHORA NICOTIANAE INFECTION. ......................................................................................... 56 Abstract .................................................................................................. 56 Introduction ............................................................................................ 57 Materials & Methods ............................................................................. 61 Results .................................................................................................... 65 Discussion .............................................................................................. 68 Conclusion ............................................................................................. 72 Literature Cited ...................................................................................... 78

vii

LIST OF TABLES

Table Page 2.1 Media components used in treatments in this study. All

treatments contained 70% growing media and 30% sterilized perlite. Each P. indica treatment contained different percentages of colonized perlite….………………

28

2.2 Cutting propagation and harvest date for the ten cultivars in eight genera grown for this study ..………………………..

29

2.3 The effect of P. indica inoculation rates on root fresh mass and percentage of rooted cuttings at harvest of six species. The hormone control was Hormodin 1 applied as a powder to the base of the cutting stem…..…………………………

30

3.1 Time in propagation, weeks until harvest and harvest months of species and individual trials…………………….

49

3.2 Nutrient analysis of fertilizer solutions used in P and N experiments…………………………………………………

50

3.3 The effect of P. indica inoculation rates on Calibrachoa × hybrida ‘Noa Yellow’, Petunia × hybrida ‘Black Velvet’, and Impatiens × hybrida ‘Sunpatiens Compact Lilac’ growth and development when supplied with no phosphorus in the fertilizer solution…..………………….

51

3.4 The effect of P. indica inoculation rates on Calibrachoa × hybrida ‘Noa Yellow’, Petunia × hybrida ‘Black Velvet’, and Impatiens × hybrida ‘Sunpatiens Compact Lilac’ growth and development when supplied with a low (9 ppm) nitrogen concentration in the fertilizer solution…...

52

4.1 The effects of three rates of Piriformospora indica and label rates of five biological control products on the production of flowers by Petunia grandiflora ‘Blue Morn,’ Petunia multiflora ‘Madness Plum,’ and Petunia × hybrida ‘Potunia Plus Purple’ that were grown in a greenhouse for 24 days……………………….……….

74

viii

LIST OF TABLES (CONTINUED)

Page 4.2 The effects of three rates of Piriformospora indica and

label rates of five biological control products on shoot dry weight by Petunia grandiflora ‘Blue Morn,’ Petunia multiflora ‘Madness Plum,’ and Petunia × hybrida ‘Potunia Plus Purple’ that were grown in a greenhouse for 24 days……………………………………………..……….

75

4.3 The effects of three rates of Piriformospora indica and label rates of five biological control products on root dry weight by Petunia grandiflora ‘Blue Morn,’ Petunia multiflora ‘Madness Plum,’ and Petunia × hybrida ‘Potunia Plus Purple’ that were grown in a greenhouse for 24 days……………………………………………………..

76

ix

LIST OF FIGURES

Figure Page 1.1 P. indica radial growth on PDA at different temperatures

12 days after inoculation. Plates were incubated in the dark. Data points represent means of six repetitions and two trials. Vertical bars on data points represent ±SE……………………………..………………………….

11

1.2 Radial growth of P. indica on potato dextrose agar at three concentrations measured 12 days after inoculation. Plates were incubated in the dark at 25°C. Data points represent means of six repetitions and two trials. Vertical bars on data points represent ±SE. …………………….………….

12

1.3 Growth of P. indica at three potato dextrose broth concentrations, measured by dry weight (A) and chlamydospore production (B). Cultures were grown at 150 RPM in the dark at 25°C and growth was measured 21 days after inoculation. Data points represent means of six repetitions and two trials. Vertical bars on data points represent ±SE………………….………………………….

13

4.1 Disease progress for 18 days after Petunia grandiflora (A),

Petunia multiflora (B), and Petunia × hybrida (C) plants were inoculated with Phytophthora nicotianae. Evaluations of disease severity were conducted every two days after inoculation (DAI) using a 0 to 3 scale based on percentage of the foliage that exhibited symptoms: 0=0%, 1=1-45%, 2=46-69%, 3=70-100%. Each range of degree severity was averaged for statistical analysis. Data were analyzed by 2-way analysis of variance, and means were separated with Fisher’s protected least significant difference (LSD) with P = 0.05. LSD values are represented by the bars above the graph…..……………

77

x

CHAPTER ONE

GROWTH OPTIMIZATION OF AN ARBUSCULAR MYCORRHIZAL-LIKE FUNGUS, PIRIFORMOSPORA INDICA

Abstract

Piriformospora indica is an arbuscular mycorrhizal (AM) -like fungus that has

been shown to provide benefits to plant symbionts by increasing nutrient uptake, disease

resistance, biomass production, and flower number. Experiments were conducted to

determine the optimal environmental conditions for growing P. indica in pure culture for

subsequent plant inoculum preparation. The effect of temperature, photosynthetic photon

flux (PPF), and potato dextrose agar (PDA) concentration on P. indica radial mycelial

growth was measured. In addition, the effect of agitation and potato dextrose broth

(PDB) concentration on P. indica mycelial mass and chlamydospore production was

examined. The optimal conditions for mycelial growth in agar occurred at PDA

concentrations of 12 and 24 g/L and temperatures ranging from 25 to 35°C. PPF had no

significant effects on mycelial growth or spore production. Increasing PDB

concentration in liquid culture resulted in an increase in fungus dry weight and a decrease

in spore production. Optimal spore production in liquid culture occurred at an agitation

speed from 150 and 200 rotations per minute (RPM) in a PDB concentration of 12 to

24 g/L. This work provides recommended guidelines for mycelial growth and spore

production of P. indica in agar and liquid culture.

Introduction

Piriformospora indica is an arbuscular mycorrhizal (AM) -like fungus that is able

to increase nutrient uptake (Gosal et al., 2010), disease resistance (Deshmukh and Kogel,

2007), and biomass production (Baldi et al, 2010) in plants. P. indica was originally

isolated from the Thar Desert in India. It is classified as a special root endophyte, which

resides only in the roots whereas endophytic fungi may occur throughout the plant (Yang

et al., 2013). Although technically an endophyte, P. indica is commonly referred to as an

AM-like plant symbiont. But unlike AM fungi, P. indica can be grown in pure culture

without a plant host. All AM fungi are from the phylum Glomeromycota whereas

endophytic species occupy all phyla of the Kingdom Fungi. Although P. indica is a

Basidiomycota it does express similar benefits to plants as the Glomeromycota. P. indica

also colonizes plants that AM species cannot, such as members of the Brassicaceae

(Deshmukh, 2006).

Because of the characteristics mentioned above, P. indica has been studied as a

soil additive, similar to mycorrhizal products. In order for a biological product to be

produced commercially, inoculum preparation must be optimized. Mass production of

P. indica is relatively simple, compared to AM fungi, since it does not require a plant

host. AM fungi are commercially produced by inoculating plant roots, allowing the

plants to grow and be colonized, and finally harvesting the roots and surrounding

substrate (Schenck, 1982). Substrate and plant roots containing hyphae and spores

become the commercial inoculum that is dried prior to being packaged and transported.

Because this process is not sterile, there is a great opportunity for contamination.

2

P. indica can be grown sterile on agar and in liquid culture with a wide range of nutrient

types including potato and tomato dextrose agar, Minimal Medium, Moser B medium,

Malt-Yeast Extract, and others (Pham et al., 2004, Verma et al., 1998). Potato dextrose

agar (PDA) and potato dextrose broth (PDB) was selected for the current experiment

because of the wide availability of the media and positive results from previous work

producing a biological control fungus in agar and liquid culture (McQuilken et al., 1997).

P. indica only produces mycelium and asexual chlamydospores, asexual spores

enclosed in a thick cell wall allowing them to withstand harsh environmental conditions,

with no known sexual stage. When conditions are favorable, chlamydospores can

germinate and infect a plant host. A previous study shows that P. indica chlamydospores

can survive up to a year at room temperature (Pham et al., 2004). This long-term survival

is ideal for commercialization of a product with a long shelf-life (Pham et al., 2004;

Vieira and Barreto, 2010). P. indica grown on agar produces hyphal growth that radiates

out uniformly from the original inoculation point. Liquid culture, also called batch

fermentation (Kavanagh, 2011), produces masses of hyphal and chlamydospore pellets.

Broth liquid culture is also used to mass produce other endophytic fungal biocontrol

agents such as Pythium oligandrum (Jackson et al., 1991) and Trichoderma spp. (Jackson

et al., 1991). In previous research, inoculation of host plants by P. indica typically occurs

by the application of spores and hyphae produced in liquid culture (Andrade-Linares et

al., 2013; Franken, 2012).

To prepare liquid culture of P. indica, colonized agar pieces are transferred to

containers with a broth nutrient solution and allowed to sporulate. Both of these steps

3

need to be optimized for commercial production. The formation of the chlamydospores

in liquid culture is affected by the growing environment such as pH, nutrient form,

temperature, photosynthetic photon flux (PPF), and oxygen concentration (Khan et al.,

2011; Carreras-Villasenor et al., 2012). Studies were conducted to optimize the growing

conditions for P. indica chlamydospores on agar and broth liquid culture for plant

inoculum preparation. The specific objectives of this study were to determine 1) the

effect of photosynthetic photon flux (PPF), temperature, and PDA concentration on

mycelial P. indica growth in agar and 2) the effect of agitation speed and PDB

concentration on P. indica mycelial growth and chlamydospore production in broth.

Materials & Methods

Expt. 1 Effect of photosynthetic photon flux on P. indica radial mycelium growth on PDA

P. indica was grown in Petri dishes with the label-recommended concentration

(39 g/L) of potato dextrose agar (PDA), (Becton, Dickson, and Co., Sparks, MD) which

is comprised of 4 g of potato starch, 20 g of dextrose, and 15 g agar dissolved in 1 L of

deionized water. The mixture was sterilized for 30 min in an autoclave at 121°C, and

plated out in 8 cm diameter Petri dishes. After 15 days, a 1 cm diameter circular plug of

agar was removed from the youngest growth region and placed in the middle of a new

agar dish containing PDA (39 g/L). Petri dishes were then placed in the dark or under a

full spectrum incandescent bulb (GE Lighting, Cleveland, OH) that delivered

10 μmol·m2·s-1 for 12 h/day in a 25°C growth chamber. Fungal growth was recorded

4

every three days by measuring the radius. Three Petri dishes were used per PPF

treatment, and the experiment was repeated three times.

Expt. 2 Effect of temperature on P. indica radial mycelium growth on PDA

P. indica was grown in Petri dishes on the same agar as used in Expt. 1. After

15 days in the dark, a 1 cm diameter circular plug of agar was removed from the youngest

growth region and placed in the middle of a new Petri dish containing a new batch of

agar. The Petri dishes were then placed into growth chambers maintained at 5, 10, 15,

20, 25, 30, 35, or 40°C. Growth according to the radius was measured every three days.

Three Petri dishes were used per temperature treatment and the experiment was repeated

three times.

Expt. 3 Effect of PDA concentration on P. indica radial mycelium growth

PDA was made at a concentration of 12, 24 and 48 g/L. NaOH was added at the

rate of 9.4, 21.9 or 43.8 ml/L for the PDB concentration treatments of 12, 24 and 48 g/L,

respectively. NaOH (1 M) was added to increase agar pH to 6.2 in order to solidify the

agar as well as to keep the pH the same among concentrations (Clark, 1965). Each liter of

solution contained 15 g of dehydrated agar powder. The solutions were autoclaved for

1 h, poured into Petri dishes, and allowed to cool to room temperature. A 1 cm diameter

plug of agar was removed from the youngest growth region of a 14-d-old culture of

P. indica on an agar plate and placed in the middle of a new Petri dish containing each of

the three PDA treatments. The Petri dishes were then incubated at 25°C for 15 days.

5

Fungal growth was recorded every three days by measuring the radius. This experiment

was repeated twice with four replications per concentration treatment.

Expt. 4 Effect of agitation speed on P. indica mycelial growth and chlamydospore

production in PDB

Liquid culture was comprised of a broth made from the label-recommended

concentration (24 g/L) of potato dextrose broth (Becton, Dickson, and Co., Sparks, MD),

which contained 4 g potato starch and 20 g dextrose dissolved in 1 L of deionized water.

A 200 mL flask was filled to 160 mL with the solution, covered with aluminum foil, and

then sterilized for 1 h in an autoclave at 121°C. A 1 cm diameter circular plug of a

14-d-old P. indica was added to each flask after the flasks were cooled to room

temperature. Flasks were placed on orbital shakers (Lab Line Instruments, Inc., Melrose

Park, IL) set at 0, 100, 150, or 200 RPM for 21 days. After 21 days, flask contents were

blended with a magnetic stir bar and stirrer for 2 min to break up the fungal pellets.

Chlamydospores were counted using a hemocytometer (Hauser Scientific Improved

Neubauer Brightline, VWR Scientific, Radnor, PA). Flask contents were placed in a

weighing dish, dried in an oven, and weighed to determine fungus (mycelia and

chlamydospore) dry weight. This experiment was repeated twice with three flasks per

agitation speed treatment.

6

Expt. 5 Effect of PDB concentration on P. indica growth and chlamydospore production

in liquid culture

PDB was tested at three concentrations: 12, 24, and 48 g/ L of deionized water.

Each replicate contained 160 mL of mixture in a 200 mL flask. Flasks were covered with

aluminum foil and autoclaved for one h at 121°C. A 1 cm diameter plug of P. indica

from a 14-d-old culture was added to each treatment after the flasks cooled to room

temperature. The flasks were then placed on an orbital shaker at 150 RPM for 21 days.

After 21 days, flask contents were blended with a magnetic stir bar and stirrer for 2 min

to break up the fungal pellets. Chlamydospores were counted using a hemocytometer.

Flask contents were placed in a weighing dish, dried in an oven, and weighed to

determine fungus (mycelia and chlamydospore) dry weight. This experiment was

repeated twice with three replications per PDB concentration treatment. Data were

analyzed using Sigma Plot ver. 11.0 (Systat Software Inc., San Jose, CA).

Results & Discussion

Changes in radial growth on PDA and spore production and dry weight in PDB

were examined to assess the effect of temperature, PPF, medium concentration, and

agitation speed on P. indica. The manipulation of temperature and PDA concentration

showed significant differences in radial growth. PPF did not alter growth significantly.

Fast radial growth on PDA is ideal for the subsequent transfer to PDB where

chlamydospores can be produced for plant inoculation in liquid culture, PDB

concentration and agitation speed played a vital role in producing chlamydospores.

7

Temperature plays an important role in mycelial growth. No growth occurred on

agar measured 12 days after inoculation at 5 to 10°C, while growth increased rapidly as

temperature increased from 15 to 25°C (Fig. 1). The optimal radial growth rate of the

fungus occurred from 25 to 35°C, while little growth occurred at 40°C. These results

agree with previous research that reported the optimal temperature range for P. indica

fungal growth was 25 to 35°C on a different medium (Pham et al., 2004). A study using

PDA reported similar results from Coniothyrium minitans, a mycoparasite used in

biological control, on radial growth producing very little growth at temperatures below

15°C, peaking near 20°C, and then decreasing drastically (McQuilken et al., 1997).

PDA concentration was investigated because of the lack of literature containing

this information. Pham (2004) tested different media for P. indica growth on agar yet the

concentrations were not presented. In the current study, no differences were observed

when PDA concentration increased from 12 to 24 g/L, while growth was inhibited at

48 g/L (Fig. 2). Previous work has shown that mycelial growth was highest when

dextrose was used as the carbon source when compared to sucrose (Pham et al., 2004).

Although radial growth of P. indica on agar was affected by temperature and PDA

concentration, PPF had no effect (data not shown). The growth of some fungi, such as

conidia production of trichoderma, is stimulated by light, specifically within the blue

spectrum (Carreras-Villasenor et al., 2012). Our study, as well as Pham (2004), found

that PPF has no influence on P. indica.

Agitation was required for chlamydospore production in liquid culture. At 0 and

100 RPM, no spores were produced while agitation speeds of 150 to 200 RPM generated

8

significant spore formation (data not presented). In contrast, fungus dry weight decreased

as agitation speed increased. The treatment without agitation produced the greatest dry

weight at 2.24 g per flask although no spores were produced. Kumar (2011) produced

similar results and reported that P. indica pellets became smaller with higher spore yields

as agitation speeds went from 0 to 300 RPM. Similarly, we observed very small pellets

with many spores at 200 RPM and only one to three large pellets with no chlamydospores

at lower agitation speeds. Kumar suggested an agitation rate of 200 RPM using Kaefer

medium for spore production (Kaefer, 1977). Agitation of fungus/hyphae in liquid

culture alters the growth rate and spore formation due to increased dissolved oxygen,

nutrient dispersion, and mechanical stress (Park et al., 2002). It is desirable to produce

chlamydospores for the commercial production of P. indica since they can withstand

temperature extremes, dehydration, and then successfully produce viable hyphae when

the environment is conducive. Thus, P. indica is well-suited for commercial production

because of the potential for a long shelf life.

Spore production of P. indica in broth decreased from 200,000 to

22,000 spores/mL as PDB concentration increased from 12 to 48 g/L (Fig. 3a). In

contrast, fungal dry mass increased from 6 to 8 g per flask as PDB concentration

increased from 12 to 48 g/L (Fig. 3b). Carbon depletion has been proposed as the cue for

P. indica sporulation (Kumar et al., 2011). Carbon becomes limited faster in liquid

culture at low carbohydrate concentrations, resulting in an increase in sporulation while

mycelial growth predominates when there is sufficient carbon available (Hood and Shew,

1997; Monteiro et al., 2005; Onilude et al., 2013).

9

Conclusion

Protocols were developed to optimize mycelial growth in agar and spore

production in liquid culture. Fast radial growth on PDA is ideal for the subsequent

transfer to PDB where chlamydospores can be produced for plant inoculation. P. indica

should be incubated between 25 to 35°C on PDA at a concentration of 12 to 24 g/L for

optimal mycelial growth. For optimal spore production in liquid culture, agitation speed

in a typical flask shaker should range from 150 to 200 RPM when using a 200 mL flask

filled to 160 mL. PDB concentrations of 12 to 24 g/L produced the highest spore counts.

These growth optimization findings will be utilized in inoculum preparation for

subsequent chapters in this dissertation.

10

y= -0.0006x3+0.0337x2-0.399x+1.19 r2=0.955

Temperature (°C)

0 10 20 30 40

Rad

ial g

row

th (c

m)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Figure 1. P. indica radial growth on PDA at different temperatures measured 12 days after inoculation. Plates were incubated in the dark. Data points represent means of six repetitions and two trials. Vertical bars on data points represent ±SE.

11

y= -0.00101x2+0.0374x+1.71 r2=0.899

Potato dextrose agar (g/L)

10 20 30 40 50

Rad

ial g

row

th (c

m)

0.0

0.5

1.0

1.5

2.0

2.5

Figure 2. Radial growth of P. indica on potato dextrose agar at three concentrations measured 12 days after inoculation. Plates were incubated in the dark at 25°C. Data points represent means of six repetitions and two trials. Vertical bars on data points represent ±SE.

12

Carbohydrate concentration (g/L)

Fung

al d

ry w

eigh

t (g)

/ fla

sk

0

2

4

6

8

10

y= -95.5x2+756x+206385 r2= 0.552

Potato dextrose broth (g/L)

10 20 30 40 50

Chl

amyd

ospo

res

(spo

res/

mL)

0.0

5.0e+4

1.0e+5

1.5e+5

2.0e+5

2.5e+5

y= -5.77e(-0.0838x) +8.16 r2= 0.713

Figure 3. Growth of P. indica at three potato dextrose broth concentrations, measured by dry weight (A) and chlamydospore production (B). Cultures were grown at 150 RPM in the dark at 25°C and growth was measured 21 days after inoculation. Data points represent means of six repetitions and two trials. Vertical bars on data points represent ±SE.

A.

B.

13

Literature Cited Andrade-Linares, D., Müller, M., Varma, A., Kost, G., Oelmüller, R., 2013. Piriformospora indica: Sebacinales and Their Biotechnological Applications. Springer Publishing Company, New York. Carreras-Villasenor, N., Sanchez-Arreguint, J., Herrera-Estrella, A., 2012. Trichoderma: sensing the environment for survival and dispersal. Microbiology 158, 3-16. Clark, F., 1965. Agar-plate method for total microbial count. Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties, 1460-1466. Franken, P., 2012. The plant strengthening root endophyte Piriformospora indica: potential application and the biology behind. Appl. Microbiol. Biotechnol. 96, 1455-1464. Harrach, B., Baltruschat, H., Barna, B., Fodor, J., Kogel, K., 2013. The mutualistic fungus Piriformospora indica protects barley roots from a loss of antioxidant capacity caused by the necrotrophic pathogen Fusarium culmorum. MPMI. 26, 599-605. Hood, M., Shew, H., 1997. The influence of nutrients on development, resting hyphae and aleuriospore induction of Thielaviopsis basicola. Mycologia 89, 793-800. Jackson, A., Whipps, J., Lynch, J., 1991. Nutritional studies of four fungi with disease biocontrol potential. Enzyme Microb. Tech. 13, 456-461. Kaefer, E., 1977. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv. Genet. 19, 33-131. Kavanagh, K., 2011. Fungi: Biology and Applications, 2nd Edition. John Wiley & Sons. West Sussex, UK. Khan, S., Bagwan, N., Iqbal, M., Tamboli, R., 2011. Mass multiplication and shelf life of liquid fermented final product of Trichoderma viride in different formulations. Adv. Biores. 2, 178-182. Kumar, V., Sahai, V., Bisaria, V., 2011. High-density spore production of Piriformospora indica, a plant growth-promoting endophyte, by optimization of nutritional and cultural parameters. Bioresour. Technol. 102, 3169-3175. McQuilken, M., Budge, S., Whipps, J., 1997. Effects of culture media and environmental factors on conidial germination, pycnidial production and hyphal extension of Coniothyrium minitans. Mycol. Res. 101, 11-17.

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Monteiro, S., Clemente, J., Henriques, Gomes R., Carrondo, M., Cunha, A., 2005. A procedure for high-yield spore production by Bacillus subtilis. Biotechnol. Prog. 21, 1026-1031. Onilude, A., Adebayo-Tayo, B., Abiodun A., Odeniyi, O., Banjo, D., Oluwaseun, E., 2013. Comparative mycelial and spore yield by Trichoderma viride in batch and fed-batch cultures. Ann. Microbiol. 63, 547-553. Park, J., Kim, YM., Kim, SW., Hwang, H., Cho, Y., Lee, Y., Song, C., Yun, J., 2002. Effect of agitation intensity on the exo-biopolymer production and mycelial morphology in Cordyceps militaris. Lett Appl Microbiol. 34, 433-438. Peškan-Berghöfer, T., Shahollari, B., Giong, P. H., Hehl, S., Markert, C., Blanke, V., Kost, G., Varma, A., and Oelmüller, R., 2004. Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant-microbe interactions and involves early plant protein modifications in the endoplasmic reticulum and at the plasma membrane. Physiol. Plant. 122, 465-477. Pham, G., Humari, R., Singh, A., Malla, R., Prasad, R., Sachdev, M., Kaldorf, M., Buscot, F., Oelmüller, R., Hampp, R., Saxena, A., Rexer, K., Kost, G., Varma, A., 2004. Plant Surface Microbiology. Springer Publishing Company, New York. Qiang, X., Weiss, M., Kogel, K.-H., and Schäfer, P., 2012. Piriformospora indica—A mutualistic basidiomycete with an exceptionally large plant host range. Mol. Plant Pathol. 13, 508-518. Rai, M., Acharya, D., Singh, A., Varma, A., 2001. Positive growth responses of the medicinal plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial. Mycorrhiza 11, 123-128. Schenck N., 1982. Methods and Principles of Mycorrhizal Research. American Phytopathological Society, St. Paul. Vieira, B., Barreto, R., 2010. Liquid culture production of chlamydospores of Lewia chlamidosporeiformans (Ascomycota: Pleosporales), a mycoherbicide candidate for wild poinsettia. Australas. Plant Path. 39, 154-160. Verma, S., Varma, A., Rexer, K., Hassel, A., Kost, G., Sarbhoy, A., Bisen, P., Bütehorn, B., Franken, P., 1998. Piriformospora indica, gen. et sp. nov., a new root colonizing fungus. Mycologia 95, 896-903.

15

Weiss, M., Sykorova, Z., Garnica, S., Riess, K., Martos, F., Krause, C., Oberwinkler, F., Bauer, R., and Redecker, D. 2011. Sebacinales everywhere: Previously overlooked ubiquitous fungal endophytes. PloS One 6, e16793. Published online.

16

CHAPTER TWO

EVALUATING THE POTENTIAL OF A NEW MYCORRHIZAL-LIKE FUNGUS, PIRIFORMOSPORA INDICA, TO IMPOROVE ADVENTITIOUS

ROOT FORMATION OF FLORICULTURE CROPS

Abstract

The mycorrhizal-like fungus Piriformospora indica has demonstrated potential to

enhance adventitious root formation (ARF) and increase root mass when applied to the

propagation substrate of unrooted cuttings (URCs). Experiments were conducted to

determine the effect of P. indica on ARF of six floriculture species (Euphorbia

pulcherrima ‘Champion Fire,’ ‘Premium White,’ ‘Supreme Bright Red,’ Scaevola

‘Fan Dancer,’ Crossandra infundibuliformis ‘Orange Marmalade,’ Dahlia × hybrida

‘Dahlietta Margaret,’ Lantana camara ‘Lucky Yellow,’ and Osteospermum × hybrida

‘Side Show White’). The treatments consisted of a soilless growing media that contained

5, 10, 20, or 30% colonized perlite (vol. colonized perlite/vol. growing media).

Inoculation with P. indica significantly increased the root fresh mass for one cultivar,

euphorbia ‘Supreme Bright Red,’ while the 20% colonized perlite treatment resulted in a

decrease in root fresh mass of scaevola and osteospermum. Rooting percentage of

euphorbia ‘Champion Fire’ increased at the 20% colonized perlite treatment, whereas,

dahlia increased significantly at the 5 and 20% colonized perlite treatments.

Osteospermum cuttings were negatively impacted by a P. indica treatment. Our results

demonstrate potential for P. indica usage for ARF enhancement with optimization for

most cultivars evaluated.

17

Introduction

Vegetative propagation is a method of plant reproduction that has been practiced

over 100 years (Preece, 2003 and references therein). Shoot tip cutting is a common

method for commercial propagation of ornamental plants. After a cutting is removed

from the stock plant, it must form adventitious roots in order to become a new plant.

Adventitious root formation, ARF, is a complex process regulated by environmental and

endogenous factors. Higher endogenous auxin concentrations or exogenously applied

auxin can speed up the process of ARF (Agullo-Anton et al., 2014; Went, 1938).

Although the majority of herbaceous annual species root easily from cuttings, some

species benefit from the application of exogenous auxin, i.e., rooting hormone.

Applying a rooting hormone has been shown to be an effective step in cutting

propagation and has been used since the 1930’s. Application is usually done by dipping

the base of the cutting stem into an auxin powder or solution before inserting the cutting

stem into the propagation medium (Preece, 2003).

Fungal symbionts such as arbuscular mycorrhizae (AM) and the mycorrhizal-like

fungus Piriformospora indica have been examined for their ability to enhance ARF and

increase root mass when applied to the propagation substrate of unrooted cuttings

(URCs). P. indica, special root endophyte, was originally isolated from the Thar Desert

in India. A special root endophyte resides only in the roots whereas endophytic fungi are

able to spread throughout the plant. P. indica is commonly referred to as an arbuscular

mycorrhizal-like plant symbiont although it actually is an endophyte. P. indica has

shown promise to increase nutrient uptake (Gosal et al., 2010), disease suppression

18

(Deshmukh and Kogel, 2007), and biomass production (Baldi et al, 2010). Arbuscular

mycorrhizal fungi share common attributes such as the previously mentioned of

P. indica. Dissimilar to AM fungi, P. indica can grow aseptically without a plant host.

This benefit is advantageous for mass production and commercialization. P. indica also

colonizes plants that AM fungi cannot, such as members of the Brassicaceae family

(Deshmukh, 2006). P. indica is a Basidiomycota yet expresses similar characteristics to

the Glomeromycota which contain all AM fungi yet endophytic fungi may occupy all

phyla of the Kingdom Fungi.

Mutualistic fungi, such as AM fungi and P. indica, added to propagation media

have been shown to increase root mass and root initiation in a range of plant species,

including Schefflera arboricola, Taxus x media, Rosa spp. ‘Scarlet Cupido’,

Arctostaphylos uva, Pelargonium spp., Euphorbia pulcherrima, and Adhatoda vasica

(Fatemeh and Zaynab, 2014, Druege et al., 2007; Rai et al., 2005; Scagel, 2003, 2004a,

2004b). These studies show promise that P. indica may be a new alternative to chemical

auxin application and also impart added benefits such as disease suppression or nutrient

uptake. The objective of this project was to determine the effect of P. indica-colonized

rooting substrate on ARF and root fresh mass of the unrooted cuttings of 8 species and

cultivars which are commonly propagated in the floriculture industry.

19

Materials & Methods

Inoculation Methodology

P. indica was originally obtained from Dr. Philipp Franken through the USDA–

APHIS (Application Number: P526-120104-009) as a single culture on agar medium.

P. indica was maintained in culture on sterile potato dextrose agar (PDA; 39g/L) (Becton,

Dickson and Company, Sparks, MD) in 9 cm diameter petri dishes. A 1-cm-diameter

plug from the youngest growth region of P. indica was placed in the center of each petri

dish and grown for 14 days at 21°C in the dark. For liquid culture, potato dextrose broth

(PDB; 24g/L) (Becton, Dickson and Company, Sparks, MD) was sterilized in 200 mL

flasks. Once cooled, five 1-cm-diameter plugs from the youngest growth region of

P. indica were aseptically transferred into each flask. The flasks with inoculum were

placed on an orbital shaker and maintained at 150 RPM for 21 days in the dark at 21°C.

Twenty mushroom spawn incubation bags, measuring 21 x 8.25 x 4.75 inches (Fungi

Perfecti, Olympia, WA) with a microporus filter patch, were each filled with 5 L of

perlite (S&B Industrial Minerals, Vero Beach, FL), moistened with 3 L of PDB (24g/L),

and sterilized for 1 h at 121°C. P. indica grown in PDB was blended aseptically with a

magnetic stir bar and stirrer for 30 s to break up the fungal pellets. Each bag was

inoculated with 25 mL of blended P. indica, sealed with an impulse sealer, and grown at

25°C in the dark. After 21 days, when the perlite was fully colonized, it was incorporated

into soilless media composed of peat, perlite, and starter fertilizer (Fafard Germination

Mix, Belton, SC) to function as both a source of inoculum and a media component. This

inoculation methodology will be the same in subsequent chapters of this dissertation.

20

Experimental Treatments

Treatments consisted of non-colonized media, non-colonized media plus rooting

hormone applied to the cutting (Hormodin 1; OHP, Inc., Mainland, PA), and four rates of

P. indica-colonized media. Hormodin 1 is a powder formation that contains 0.1% indole-

3-butyric acid that was applied as a dip to the bottom 1 cm of the stem prior to inserting

the cutting into the propagation media. To keep the growing medium matrix consistent

throughout the experiments, all treatments consisted of a growing media containing 30%

sterilized perlite and 70% commercial growing media measured by volume (Table 1).

This amount of perlite was used because commercial propagation media contain no more

than 30% perlite on a volume basis. Each P. indica treatment contained different

percentages of colonized perlite. For example, the 10% colonized perlite treatment

contained 10% colonized perlite, 20% sterilized non-colonized perlite, and 70% growing

media, while the 20% colonized perlite treatment contained 20% colonized perlite, 10%

sterilized non-colonized perlite, and 70% growing media. The 30% colonized perlite

treatment contained 30% colonized perlite and 70% growing media.

Treatments for each plant taxa are listed in Table 1. Euphorbia treatments

consisted of non-colonized media, non-colonized media plus rooting hormone applied to

the cutting (Hormodin 1), and media inoculations with each of three rates of P. indica

(10, 20, and 30% colonized perlite). Crossandra and osteospermum cutting treatments

consisted of non-colonized media and media inoculations with each of three rates of

P. indica (10, 20, and 30% colonized perlite). Scaevola cutting treatments consisted of

non-colonized media and media inoculations with each of three rates of P. indica (5, 10,

21

and 20% colonized perlite). Dahlia and lantana treatments consisted of non-colonized

media, non-colonized media plus rooting hormone applied to the cutting, and media

inoculations with each of three rates of P. indica (5, 10, and 20% colonized perlite).

Plant Materials

Unrooted cuttings of Euphorbia pulcherrima ‘Champion Fire,’ ‘Premium White,’

‘Supreme Bright Red’ (Dummen USA, Inc., Hillard, OH), Scaevola ‘Fan Dancer,’

Crossandra infundibuliformis ‘Orange Marmalade,’ (Ecke Ranch, Encinitas, CA), Dahlia

× hybrida ‘Dahlietta Margaret,’ and Lantana camara ‘Lucky Yellow’ (Ball Horticultural

Co., West Chicago, IL) were delivered via air freight by two-day delivery.

Osteospermum × hybrida ‘Side Show White’ unrooted cuttings were harvested from

stock plants cultivated in a glass greenhouse (Clemson University, Clemson, SC). Stock

plants were grown in soil-less media (Fafard 3B, Belton, SC) and fertilized during each

irrigation with a 200 ppm nitrogen solution using Peters Excel 15-5-15 Cal-Mag (The

Scotts’ Co., Marysville, OH). Osteospermum stock plants were grown at an average

daily temperature of 21-22°C. Cuttings produced on site were placed in a 10°C cooler for

two days to simulate commercial postharvest conditions. The propagation environment

consisted of a mist system, a retractable shade curtain that was engaged when natural

radiation exceeded 450 W/m2, heating/ventilation set points at 22/26°C, and bottom heat

that provided a growing media minimum temperature of 24.4°C. The cuttings received no

additional fertilizer beyond the nutrients in the growing media.

22

Data Collection

Cuttings were harvested on different dates depending on root initiation (Table 2).

At harvest root fresh weight and the percentage of rooted cuttings were recorded.

Subsamples of roots were collected to determine root colonization. Roots were

submerged in 10% KOH (w/v) for 3 h in a water bath at 60°C to remove any

pigmentation. Roots were then rinsed with deionized water twice and stained with 0.05%

Trypan Blue for 24 h. Roots were viewed by mounting in 50% glycerin (Phillips and

Hayman, 1970) and observed with a compound light microscope. Colonization was

confirmed to be successful by the presence of chlamydospores. Data were analyzed

using JMP ver. 10 (SAS Institute Inc., Cary, NC).

Results

P. indica inoculation of the growing media produced results that varied between

species and cultivars. Euphorbia ‘Supreme Bright Red’ cuttings increased significantly

in root mass when colonized with P. indica at the 10 and 20% perlite inoculation levels in

comparison to the non-colonized control (Table 3). Euphorbia ‘Supreme Bright Red’

with P. indica treatments had higher rooting percentages than the hormone control. Root

fresh mass of euphorbia ‘Champion Fire’ did not respond significantly to P. indica

treatments or the rooting hormone treatment. Significantly higher rooting percentages

were produced by the euphorbia ‘Champion Fire’ 20% perlite treatment as well as the

rooting hormone control. Euphorbia ‘Premium White’ did not respond to P. indica

treatments or the rooting hormone treatment.

23

Osteospermum showed a negative root growth response at the 20% colonized

perlite treatment while the 10 and 20% colonized perlite treatment had negative effects

on the rooting percentage when compared to the control. Twenty percent colonized

perlite had an adverse effect on scaevola fresh root weight, while no treatment affected

the rooting percentage (Table 3).

The P. indica treatments did not affect dahlia in terms of mean root mass,

although the plants with rooting hormone produced a significant increase (Table 3).

Rooting percentage was increased by the 5 and 20% colonized perlite treatments as well

as the rooting hormone treatment. Dahlia had the lowest rooting percentage of all the

species in this study with the non-colonized control at only 25.5% success while the

hormone control yielded 57% rooted cuttings. Application of rooting hormone on

lantana negatively affected rooting compared to the non-colonized control. Crossandra

and lantana did not significantly respond to treatments in terms of fresh root mass or

rooting percentage. All plant species and treatments inoculated with P. indica were

confirmed for inoculation by viewing subsets of root samples at the end of the

experiment.

Discussion & Conclusion

P. indica enhanced ARF and increased root mass when applied to the propagation

substrate of some ornamental URCs. In this experiment it was found that euphorbia

‘Champion Fire,’ ‘Supreme Bright Red,’ and dahlia could all potentially benefit from the

addition of P. indica inoculation in the propagation media. Similarly, Druege (2007)

24

found that P. indica promotes ARF on two of three floriculture species tested.

Pelargonium and poinsettia root number and length were enhanced, whereas petunia did

not show positive responses from inoculation. As seen in the current study, the addition

of a fungal symbiont is neither always beneficial nor harmful. Within each variety, there

was at least one level of P. indica inoculation that showed no difference when compared

to the non-hormone control. Similarly, Vosatka (1999) found that application of AM

fungi in the rooting substrate had no effect on Euphorbia pulcherrima ARF.

Auxin application has been shown to increase ARF in cuttings of most plant

species. When plants are cut for vegetative propagation they release CO2 and other

metabolites which can be perceived similar to the root exudation process. This stimulus

can cause the fungal symbiont to activate and produce chemicals conducive to ARF such

as phytohormones or compounds that decrease auxin oxidation (Scagel, 2004a;

Sirrenberg, 2007). Most research shows that communication occurs prior to colonization,

thus giving benefit to plants without roots (Douds et al., 1995, Druege et al., 2007; Felten

et al., 2010; Scagel, 2004b). In this study a commonly used commercial rooting hormone

was compared to P. indica. Similar outcomes to P. indica were observed showing that

rooting benefit of auxin was dependent on plant variety and there was not always an

increase over the control. Many researchers propose that P. indica not only produces and

excretes auxin but also upregulates auxin biosynthesis genes inside the plant cell (Dutra

et al., 1996; Felten et al., 2010; Hilbert et al., 2012; Hilbert et al., 2013; Lee et al., 2011;

Ludwig-Muller and Guther, 2007; Schafer et al., 2009; Vadassery et al., 2008). Levels of

auxin production induced by P. indica vary with plant host (Sirrenberg et al., 2007;

25

Sukumar et al., 2013; Vadassery et al., 2008). For example, it was found that auxin

levels in P. indica-colonized Chinese cabbage roots were two-fold compared to the

control, yet in arabidopsis, the auxin levels were not higher (Lee et al., 2011). However,

phenotypic responses, i.e., stunted and highly branched roots, mimicking external

application of auxin, were observed when arabidopsis was co-colonized with P. indica in

sterile culture (Sirrenberg et al., 2007). It has been documented that some plant species

and cultivars have higher auxin sensitivities, auxin type or concentration, than others

(Garrido et al., 1998). For instance, when paeonia ‘Yang Fei Chu Yu’ was dipped in

2000 mg L-1 indole-3-butyric acid, prior to sticking, the average root number and length

significantly increased whereas the use of 3000 mg L-1 indole-3-butyric acid

significantly reduced rooting (Guo et al., 2009). Other investigators also found the use of

AM fungi or AM-like fungal inoculants having inconsistent results on ARF (Verkade and

Hamilton, 1987; Scagel and Reddy, 2003, Vosatka et al., 1999).

Although it would be economically beneficial to propagate each plant, especially

within the same species, with the same cultural practices, both fungal symbiont

applications and auxin applications are often cultivar specific. We found P. indica can

promote ARF in some taxa, but inconsistencies were seen among the different

inoculation levels. When P. indica was compared to the commonly used commercial

auxin, we found it to be as good of a rooting promoter as the industry standard, and it

may also provide benefits later in the plant’s life. Early inoculation could benefit the

plant downstream through increased disease resistance (Chapter 4) or nutrient uptake

(Chapter 3). P. indica application could also provide an economic benefit in that it saves

26

time and money. Perlite, a common component of propagation media, could be

inoculated with P. indica and then incorporated into the propagation media used for

cutting establishment. This would eliminate the relatively time-consuming step of

dipping individual cuttings into a rooting hormone prior to planting. Supplementary

research is needed to determine optimum inoculation concentrations on each plant

variety.

27

Table 1. Media components used in treatments in this study. All treatments contained 70% growing media and 30% sterilized perlite. Each P. indica treatment contained different percentages of colonized perlite.

Treatment Growing media

(% volume)

Perlite (% volume) Colonized perlite Non-colonized

perlite Non-colonized control 70 0 30

Non-colonized control + Hormodin 1 70 0 30 5% colonized perlite (v/v) 70 5 25

10% colonized perlite (v/v) 70 10 20 20% colonized perlite (v/v) 70 20 10 30% colonized perlite (v/v) 70 30 0

28

Table 2. Cutting propagation and harvest date for the ten cultivars in six genera grown for this study. Plant Rep Propagate Harvest Crossandra ‘Orange Marmalade’ 1 August 8, 2012 August 28, 2012 2 October 12, 2012 November 2, 2012 Dahlia ‘Dahlietta Margaret’ 1 April 26, 2012 May 29, 2012 2 May 2, 2012 June 1, 2012 Euphorbia ‘Champion Fire’ 1 August 7, 2012 August 29, 2012 2 September 11, 2012 October 2, 2012 Euphorbia ‘Premium White’ 1 August 7, 2012 August 24, 2012 2 March 26, 2013 April 15, 2013 Euphorbia ‘Supreme Bright Red’ 1 August 7, 2012 August 22, 2012 2 March 26, 2013 April 15, 2013 Lantana ‘Lucky Yellow’ 1 April 26, 2012 May 10, 2012 2 May 2, 2012 May 18, 2012 Osteospermum ‘Side Show White’ 1 February 12, 2012 March 3, 2012 2 April 9, 2013 April 30, 2013 Scaevola ‘Fan Dancer’ 1 March 21, 2012 April 6, 2012 2 April 2, 2012 April 20, 2012

29

Table 3. The effect of P. indica inoculation rates on root fresh mass and percentage of rooted cuttings at harvest of six species. The hormone control was Hormodin 1 applied as a powder to the base of the cutting stem.

Z same lettering within rows indicate no significant differences between treatments (Tukey’s HSD 5% level). Dashes indicate data not taken.

Species

Cultivar

Non- colonized

control

Hormone control

P. indica-colonized perlite (% volume)

5 10 20 30

Root fresh mass (g)

Euphorbia Champion Fire 0.255 az 0.233 a - 0.265 a 0.305 a 0.231 a

Premium White 0.381 a 0.439 a - 0.398 a 0.400 a 0.417 a

Supreme Bright Red 0.286 b 0.392 ab - 0.417 a 0.452 a 0.383 ab

Crossandra Orange Marmalade 0.299 a - - 0.310 a 0.299 a 0.276 a

Osteospermum Side Show White 0.157 a - - 0.156 a 0.046 b 0.196 a

Scaevola Fan Dancer 0.164 a - 0.126 ab 0.141 a 0.082 b -

Dahlia Dahliette Margaret 0.055 b 0.187 a 0.056 b 0.053 b 0.060 b -

Lantana Lucky Yellow 0.159 ab 0.106 c 0.190 a 0.164 ab 0.140 bc -

Cutting rooting (%)

Euphorbia Champion Fire 79 b 96 a - 95 ab 97 a 81 ab

Premium White 89 a 92 a - 91 a 89 a 96 a

Supreme Bright Red 89 ab 82 b - 98 a 95 a 97 a

Crossandra Orange Marmalade 100 a - - 100 a 100 a 100 a

Osteospermum Side Show White 86 a - - 72 b 46 c 74 ab

Scaevola Fan Dancer 82 a - 86 a 82 a 76 a -

Dahlia Dahliette Margaret 25 c 57 a 42 ab 35 bc 44 ab -

Lantana Lucky Yellow 98 a 95 a 97 a 95 a 98 a -

30

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34

CHAPTER THREE

RESPONSE OF FLORICULTURE CROPS TO NITROGEN AND PHOSPHORUS RESTRICTION WHEN COLONIZED WITH THE MYCORRHIZAL-LIKE

FUNGUS, PIRIFORMOSPORA INDICA

Abstract

Piriformospora indica is a fungal endophyte that has been shown to provide

benefits to plant symbionts by increasing nutrient uptake, disease resistance, biomass

production, and flower number. Experiments were conducted to determine the effect of

P. indica on nitrogen (N) and phosphorus (P) uptake by calibrachoa, impatiens, and

petunia. The treatments consisted of a soilless growing media that contained 0, 10, 20, or

30% colonized perlite (vol. colonized perlite/vol. growing media). Calibrachoa grown at

a low N concentration significantly increased in flower number, shoot and root growth at

the 20 and 30% colonized perlite treatments. Petunia and impatiens grown at a low N

concentration exhibited an increase in shoot dry weight and flower number at the 30%

colonized perlite treatment. When P was withheld from the fertilizer solution, calibrachoa

showed an increase in shoot and root growth and flower number at the 10% colonized

perlite treatment. Impatiens grown without P showed a reduction in flower number and

plant height at the 20 and 30% colonized perlite treatments. Our results suggest that

inoculation with P. indica can enhance N uptake, biomass, and flower number of the

three floriculture species tested when N was restricted, whereas only calibrachoa

responded positively when P was restricted.

35

Introduction

Plant nutrition is an important component in growing greenhouse crops. Proper

ratios as well as concentrations of certain nutrients are crucial to produce commercial

quality plants. An essential plant nutrient is defined as one that is an intrinsic component

in the structure or metabolism of a plant or whose absence causes severe abnormalities in

plant growth, development, or reproduction (Taiz, 2006). Nutrient uptake is regulated by

environmental factors such as pH, temperature, and moisture (Miransari, 2011). In

addition, uptake can be enhanced in nature by soil microflora, specifically beneficial

fungi (Javot et al., 2007).

Mutualistic symbiotic relationships are formed between plants and

microorganisms in order to optimize survival against biotic and abiotic factors.

Symbiosis is the association and coexistence of two unlike organisms. Fungi and bacteria

can form symbiotic relationships with plants and animals which can be categorized as

parasitism, mutualism, or commensalism (Hamilton et al., 2012). A mutualistic union is

beneficial to both organisms. Fungi and bacteria form associations with plant roots and

benefit the plant through nutrient fixation, nutrient uptake, disease suppression, and

drought resistance (Barea et al., 2005; Jeffries et al., 2003; Raaijmakers et al., 2009).

Bacteria can fix nitrogen (N) and fungi aid in the mineralization of P (Krey et al., 2011).

The microorganism benefits from the union by receiving fixed carbon and a protective

residence (Pfeffer et al., 1999).

Fungal symbionts, such as arbuscular mycorrhizae (AM) and the mycorrhizal-like

fungus Piriformospora indica, have demonstrated potential to increase nutrient uptake.

36

The special root endophyte, P. indica, was originally isolated from the Thar Desert in

India. A special root endophyte resides only in the roots whereas endophytic fungi are

able to spread throughout the plant (Yang et al., 2013). Though technically an

endophyte, P. indica is commonly referred to as an arbuscular mycorrhizal-like plant

symbiont. P. indica and arbuscular mycorrhizal can increase nutrient uptake (Gosal et

al., 2010) disease suppression (Deshmukh and Kogel, 2007), and biomass production

(Baldi et al, 2010). In addition, P. indica can colonize plants that arbuscular mycorrhizae

cannot, such as members of the Brassicaceae family (Deshmukh, 2006). Dissimilar to

AM fungi, P. indica is able to be grown aseptically without a plant host which is

advantageous for mass production and commercialization. Endophytic fungi occupy all

phyla of from the Kingdom Fungi and all AM fungi are only from the phyla

Glomeromycota. P. indica expresses similar characteristics to Glomeromycota yet it is a

Basidiomycota.

The essential plant nutrient N is found at the highest concentration in plant tissue

of all macronutrients. N is an important component of many structural, genetic, and

metabolic compounds in plant cells such as amino acids, proteins, and nucleic acids. N-

deficient plants usually are smaller and show chlorosis in the older lower leaves.

Yellowing is usually not shown in younger leaves until the deficiency is extreme due to

the mobility of the element. Nitrogen can only be taken up by plants in the following

forms: ammonium (NH4+), nitrate (NO3), nitrite (NO2

-), organic nitrogen, and molecular

nitrogen. The majority of plants absorb N from the growing media as nitrate yet it must

be reduced to ammonia (NH3) before it can be assimilated by the plant. This process

37

consumes energy and requires enzyme producing microorganisms. Inorganic fertilizers,

nitrate and ammonium, both must be converted before use by the plant. One such way is

through transfer between a symbiotic fungi and a plant host (Hairu, 2012; Read, 2003).

Between 30% and 42% of the N taken up by plants can be transferred by AM fungi

(Govindarajulu, et al., 2005; Mader, et al., 2000). Multiple forms of N, such as

ammonium, nitrate, and amino acids, are taken up and assimilated to the host plant (Jin et

al., 2005). Jin discovered the predominate free amino acid was arginine in the

extraradical mycelium of an arbuscular mycorrhizae grown in vitro with carrot roots. N

is rapidly converted to this amino acid suggesting an avoidance of toxic ammonium. In

this experiment over half of the total N transferred to the host was in the form of arginine,

a polyphosphate. Arginine can then be transferred across to the plant and released as

ammonium. N is also capable of being transferred to the plant host without a carbon

transfer (Cruz et al., 2007; Govindarajulu et al., 2005).

Phosphorus (P) is also an essential plant nutrient and a non-renewable resource. It

is estimated that by 2100 40-60% of the world’s P supply could be extracted (Van

Vuuren et al., 2010). In the plant, P is involved in the makeup of phospholipids, DNA

and RNA, starch synthesis, transport of sugars across the chloroplast membrane, energy

metabolism and ATP production (Ripley et al., 2004). Because of the importance P plays

in energy storage and structural integrity, deficiencies can greatly impact growth,

formation, and overall function of the plant (Taiz, 2006). Deficient plants usually are

stunted with dark green or purple coloration and possible necrosis. P can be found in the

soil in organic and inorganic forms. Inorganic P can quickly become fixed and useless

38

because of its natural negatively charged state. P reacts with positively charged iron,

aluminum, and calcium ions to form a complex which cannot be taken up easily by the

plant. Soil pH plays a major role in absorption of P. P ions reach the plant through root

interception, mass flow, and diffusion (Bolan, 1991). Organic forms are found in humus

and organic material whereas inorganic phosphorus (Pi) is freely available for absorption

(Ciereszko and Zebrowska, 2011). Plants can adapt to increase the absorption of organic

P through processes such as increased organic acid production and exudation, increased

phosphate transporters, or by the union with microorganisms such as AM fungi (Deressa

and Schenk, 2008; Ciereszko and Zebrowska, 2011). Enhancement is due to faster

movement of P, larger soil exploration area, and increased soil P solubilization (Bolan,

1991). Bolan (1991) found that the concentration inside the hyphae is 1000 times greater

than that in the surrounding soil proving that P must be taken up against an

electrochemical potential. In contrast, it was suggested that growth of mycorrhizal-

associated plants can decrease because of the carbon transfer. Approximately 20% of the

carbon assimilated by the host plant can be sent to the fungal symbiont (Finlay and

Soderstrom, 1992; Parniske, 2008). P transfer to the host is not dependent on the plant

demand for P but by the flux of carbohydrates back to the fungus. The amount of P

contributed from the fungus to the plant varies between host and fungal symbiont and can

range from a very small percentage to the majority of the accumulated P (Javot et al.,

2007).

Both mycorrhizae and fungal endophytes contribute to enhanced nutrient uptake

by increasing surface area, solubilization, and nutrient transport across fungal walls

39

(Bolan, 1991). Studies have shown that both AM fungi and P. indica can enhance N, P,

Cu, and Zn uptake (Bucking et al., 2007; Franken, 2012; Javaid, 2009; Rutto et al., 2002).

Much research has been performed on the effects of arbuscular mycorrhizae nutrient

enhancement on plants ranging from sugar beet to marigolds yet much less work has

focused on P. indica, specifically in regards to ornamental plants (Bagyaraj and Powell,

1985, Hairu et al., 2012, Javaid, 2009, Koegel et al., 2013, Terry and Ulrich, 1973,

Miransari, 2011). The objective of this project was to determine the effect of P .indica

on plant growth in a nitrogen and phosphorus-restricted growing medium. Specifically,

flower number, shoot and root dry weight, and height were measured on calibrachoa,

impatiens, and petunia, while the plants are supplied insufficient concentrations of N or

P.

Materials & Methods

Calibrachoa × hybrida ‘Noa Yellow,’ Impatiens × hybrida ‘Sunpatiens Compact

Lilac,’ (Ecke Ranch, Encinitas, CA), and Petunia × hybrida ‘Black Velvet’ (Ball

Horticultural Co., West Chicago, IL) unrooted cuttings were delivered via air freight by

two-day delivery. Cuttings were propagated in trays containing 105 individual cells

(25 cm3/cell). The cuttings received no fertilizer beyond the nutrients supplied in the

propagation media (Fafard Germination Mix, Belton, SC) which had a pH of 6.1, an

electrical conductivity of 0.86 mS/cm, and a starter charge of nutrients that included N, P,

K, Mg and Ca at 29, 8, 48, 55, and 47 ppm, respectively. Propagation of unrooted

cuttings was carried out in a greenhouse using intermittent mist. Shade curtains (50%)

40

were engaged when the irradiance measured outside the greenhouse exceeded 450 W/m2.

Heating/ventilation set points were 22/26°C and no bottom heat was used. Propagation

dates varied with species (Table 1). P. indica inoculum preparation was identical to

chapter 2 (see inoculation methodology section).

Experimental treatments consisted of a non-colonized media and media colonized

with each of three rates of P. indica. All media treatments were comprised of 70% (by

volume) commercial media (Fafard 3B without starter charge, SunGrow, Belton, SC)

mixed with an additional 30% perlite (by volume). The perlite fraction was comprised of

a mixture of colonized and non-colonized perlite. The colonized perlite was 0, 33, 66 or

100% of the total added perlite (by volume). These four inoculation treatments are

referred to in this manuscript by the percentage of the total media volume that was

comprised of P. indica-colonized perlite, e.g., 0, 10, 20 and 30%. Colonized perlite was

incorporated into the growing media and a water soluble fertilizer (20-10-20, J.R. Peters

Laboratory, Allentown, PA) was incorporated to the growing media at the rate of

296.5 g/m3.

Each of the four media treatments consisted of 40 containers. Twenty containers

per media treatment were placed in a block on the vent side of the greenhouse, while the

other 20 containers per treatment were placed in a block on the fan side of the

greenhouse. Thus, each block consisted of 4 treatments containing 20 plants each, and the

treatments were arranged in a random pattern within each block. The 20 containers used

per media treatment were placed in a pot-to-pot arrangement with 90 cm between the

treatments in order to reduce the potential for cross-contamination. The P and N

41

experiments began at the time of transplant, and the transplant dates varied according to

species (Table 1). Data were analyzed using JMP ver. 10 (SAS Institute Inc., Cary, NC).

Expt. 1. Effect of P. indica inoculation on plant growth and flowering in a phosphorus-

limited environment

Rooted cuttings were transplanted into the four media treatments at the start of the

experiment into 12.7 cm diameter pots (Landmark Plastic Corporation, Akron, OH). The

growing media contained 29 ppm N and 8 ppm P with an electrical conductivity of

0.86 mS/cm. Plants were treated at transplant with a fertilizer solution containing all

essential nutrients except P (Table 2). The fertilizer solution consisted of the following

components: 1.5 mmol/L Ca(NO3)2, 2.7 mmol/L MgSO4, 2.2 mmol/L KNO3, and

Greencare Minors (2.7 μmol/L Iron EDTA, 3.3 μmol/L MnSO4, 3.1 μmol/L ZnSO4,

1.6 μmol/L CuSO4, 4.0 μmol/L H3BO3, 0.5 μmol/L NaMoO4) (The Blackmore Co.,

Belleville, MI). The fertilizer solution was mixed in a 100 gallon container and pumped

to plants through pressure regulated, drip emitters.

Expt. 2. Effect of P. indica inoculation on plant growth and flowering in a nitrogen-

limited environment

Rooted cuttings were transplanted into 12.7 cm diameter pots with soilless media

containing 29 ppm N and 8 ppm P with an electrical conductivity of 0.86 mS/cm. Plants

were treated at transplant with a fertilizer solution containing all essential nutrients and a

low concentration (9 ppm) of N (Table 2). The fertilizer solution delivered was

42

comprised of the following: 3 mmol/L CaNO3 , 2.7 mmol/L MgSO4, 22 mmol/L KNO3,

17 mmol/L CaCl2 · 2H2O, and the same Greencare Minors as Expt. 1. Inoculation

protocols, media treatments, and experimental design were the same as Expt. 1.

Data Collection

At harvest, plants were evaluated for root and shoot fresh and dry weight as well

as final flower number. Ten young, fully emerged leaves were selected for leaf

nutritional analysis. Random leaves were removed from each of the twenty container

treatment blocks, dried, and measured using the Inductively Coupled Plasma Mass

Spectrometer (ICP-MS) at USDA-ARS Toledo, OH. Total plant height was only

recorded for Impatiens. When roots were harvested for root weight data collection,

700 mL of media in total was collected from all twenty containers in each treatment

block. Media samples were then analyzed for electrical conductivity, pH, phosphorus,

potassium, calcium, magnesium, and nitrate concentrations were measured (Clemson

University Soil Testing Facility).

At harvest, roots were sampled to determine root P. indica colonization by the

presence of chlamydospores. Roots were first submerged with 10% w/v KOH for 3 h in a

water bath at 60°C to remove any pigmentation. Roots were then rinsed twice with

deionized water and stained with Trypan Blue for 24 h. The stain was prepared by

mixing equal volumes of water, glycerin, and lactic acid to 0.05% Trypan Blue. Roots

were viewed by mounting in 50% glycerin (Phillips and Hayman, 1970) and examined

with a compound microscope.

43

Results

At harvest, the growing media for all species contained 0 ppm P. Percentage dry

weight of P in leaves measured at the end of the experiment was low for all species and

very similar among treatments: Calibrachoa (0.057-0.063), petunia (0.011-0.015), and

impatiens (0.09-0.13).

Calibrachoa showed significant increases in shoot dry weight and flowering at the

10% colonized perlite treatment (Table 3). Flower number was enhanced by 39% and

shoot dry weight by 37% at the 10% colonized perlite treatment when compared to the

control. Root dry weight was not significantly different than other treatments.

Inoculation treatments in petunia growing media did not yield enhanced growth

parameters when P was withheld from the fertilizer solution. The only significant

treatment effect was that shoot dry weight was increased at the 30% colonized perlite

treatment (Table 3). All remaining growth parameters were not significantly affected by

the addition of P. indica.

Impatiens only showed significant responses for plant height at the 10% colonized

perlite treatment when supplied with no P (Table 3). Flower number and plant height

significantly decreased in 20 and 30% colonized perlite treatments when compared to the

control. All other parameters were not affected by P. indica inoculation.

At harvest, the growing media for all species contained 0 ppm N. Percentage dry

weight of N in leaves measured at the end of the experiment was low for all species and

similar amongst treatments. Petunia contained a range of 2.4 to 2.7 percent dry weight of

N. Calibrachoa leaf N nutritional values for the P. indica treatments ranged from 1.23-

44

1.53% dry weight where the control was 1.08% dry weight. Impatiens leaf N nutritional

values for the P. indica treatments ranged from 1.55-1.65% dry weight where the control

was 1.40% dry weight. This suggests that P. indica inoculation improved N uptake.

Petunia, calibrachoa, and impatiens consistently showed treatment responses at

the higher P. indica inoculation levels. Calibrachoa had significant increases on all

growth and flowering parameters at the 20 and 30% colonized perlite treatments when

compared to the control and the 10% colonized perlite treatments (Table 4). Flower

number was enhanced by 79% at the 20 and 30% colonized perlite treatments when

compared to the control.

Petunia showed significant growth and flowering responses at the 30% colonized

perlite treatment (Table 4). Flower number, shoot dry weight, and total height was

enhanced by 30% colonized perlite treatments when compared to the control. Root dry

weight was not affected by the P. indica treatments.

Similar to petunia, impatiens only showed significant responses at the

30% colonized perlite treatment (Table 4). Flower number and shoot dry weight was

significantly enhanced by 30% colonized perlite treatments when compared to the

control. Root fresh and dry weight was not affected by P. indica treatments. The 30%

colonized perlite treatment was significantly highest with a total growth of 27.9 cm. The

10 and 20% colonized perlite treatment were not significantly different than the control at

any parameter. All plant species and treatments inoculated with P. indica were

confirmed for inoculation by viewing root samples at the end of the experiment.

45

Discussion & Conclusion

While supplying suboptimal amounts of N, P. indica was able to increase flower

number and dry weights of roots and shoots on three floriculture species. Calibrachoa

grown at a low N concentration significantly increased in flower number, shoot and root

growth at the 20 and 30% colonized perlite treatments. Petunia and impatiens grown at a

low N concentration exhibited an increase in shoot dry weight and flower numbers at the

30% colonized perlite treatment. Studies show that beneficial fungi can uptake multiple

forms of N as well as transport assimilated N to plant roots (Hairu, et al., 2012; He, et al.,

2003). Research suggests that growth promotion of the host plant by P. indica is

accompanied by a co-regulated stimulation of enzymes involved in nitrate metabolism.

When tested in sterile culture, large amounts of N were moved to the aerial parts of the

plants versus the control, suggesting that a key enzyme, NADH-dependent nitrate

reductase, is stimulated by the presence of the fungi (Sherameti, 2005). Although the

plant may not benefit from a fungal symbiont, N will be taken up by the fungus shown by

the transfer of radioactively marked N (Javot et al., 2007). In the current study, it was

found that P. indica has the ability to improve growth parameters at higher (20 & 30%

colonized perlite) levels presumably due to and increase N uptake. Other studies also

show an increase of N uptake by fungi on celery, wheat, corn, barley, and root organ

cultures (Ames et al., 1983; Fellbaum et al., 2012; Achatz, 2010; Hawkins et al., 2000).

Though many studies prove the benefit of fungi on P uptake, our study did not

show consistent improvement in P uptake, expressed by growth benefits, in the colonized

perlite treatments. It is possible that all of the P provided in the starter charge was rapidly

46

used for plant growth prior to colonization. Thus, the ability of the fungus to help

solubilize organic P sources to the point of relieving deficiency was outweighed by

damage done by the efflux of carbon. It would be necessary to test different levels of P

to avoid fertilizer induced pathogenicity from the inoculation with P. indica (Shamshiri et

al., 2011). In contrast to this theory, Achatz found that P concentration supplied was

irrelevant to P uptake, change in biomass, and grain production on P. indica inoculated

Hordeum vulgare (Achatz et al., 2010). In a similar study, when Cicer arietinum L. was

inoculated with P. indica and grown at a low concentration of P, dry biomass as well as

percentage P was not increased at harvest (Meena and Mesapogu, 2010). In the current

study, petunia plants at the highest level of P. indica inoculation increased shoot dry

weight by 22% (Table 3). Kumar revealed similar results in that maize biomass was

increased 2.5 fold when inoculated with P. indica when deprived of P (Kumar et al.

2011). In addition, several studies have shown that when petunia was supplied with a low

amount of P the plants inoculated with AM fungi consistently had higher biomass as well

as flower number (Gaur et al., 2000; Shamshiri et al., 2011). More studies are required to

determine if P. indica can be beneficial in the uptake at P when supplying suboptimal

amounts versus completely withholding P. In conclusion, we found that the application

of P. indica on floriculture crops increased N uptake when N was limited, as shown by an

increase in growth; however, P uptake did not appear to be enhanced as observed by

inconsistencies in growth. Because P. indica can increase the uptake of N, it is possible

less N could be supplied during fertilization.

47

Table 1. Timing information for the individual trials conducted on each of three species for the phosphorus (Expt. 1) and nitrogen restriction (Expt. 2) experiments.

Time period of trials Expt. Species Days in propagation Weeks to

harvest 1 2

-P Impatiens 21 7 October-12 August-13 Calibrachoa 35 9 February-13 August-13

Petunia 21 10 April-12 October-13 -N Impatiens 21 7 June-13 August-13

Calibrachoa 35 8 March-13 August-13 Petunia 21 6 March-13 June-13

48

Table 2. Nutrient analysis of fertilizer solutions used in phosphorus (Expt. 1) and N (Expt. 2) experiments.

Nutrient

Experiment -P -N

(ppm) N 55 9 P 0.2 24 K 101.4 111.2 Ca 53.6 106.2 Mg 34.2 36.4 S 48 68 Fe 0.91 0.02 Zn 0.53 0.64 Mn 0.58 0.56 Cu 0.26 0.26 B 0.24 0.29 Cl 15.1 194

EC

1.0 (mS/cm)

1.22 pH 7.1 6.4

49

Table 3. The effect of P. indica inoculation rates on Calibrachoa × hybrida ‘Noa Yellow’, Petunia × hybrida ‘Black Velvet’, and Impatiens × hybrida ‘Sunpatiens Compact Lilac’ growth and development when supplied with no phosphorus in the fertilizer solution (Expt. 1).

Colonized perlite (% volume)

Flower number

Shoot dry weight (g)

Root dry weight (g)

Total height increase (cm)

Calibrachoa 0 7.0 bc Z 2.7 c 2.2 a - 10 11.4 a 4.3 a 3.1 a - 20 6.8 c 2.9 c 2.4 a - 30 9.4 ab 3.7 b 1.9 a -

Petunia 0 24.2 ab 4.3 b 1.4 a - 10 23.7 ab 4.5 ab 1.5 a - 20 21.2 b 4.3 b 1.5 a - 30 29.3 a 5.5 a 1.5 a -

Impatiens 0 13.7 a 5.4 a 5.3 a 5.9 b 10 14.1 a 6.0 a 6.4 a 7.0 a 20 9.2 b 4.3 a 5.6 a 4.9 c 30 7.2 b 4.1 a 5.4 a 5.1 c

Z same lettering within columns indicate no significant differences between treatments (Tukey’s HSD 5% level). Dashes indicate data not taken.

50

Table 4. The effect of P. indica inoculation rates on Calibrachoa × hybrida ‘Noa Yellow’, Petunia × hybrida ‘Black Velvet’, and Impatiens × hybrida ‘Sunpatiens Compact Lilac’ growth and development when supplied with a low (9 ppm) nitrogen concentration in the fertilizer solution (Expt. 2).

Colonized perlite (% volume)

Flower number

Shoot dry weight (g)

Root dry weight (g)

Total height increase (cm)

Calibrachoa 0 0.7 b Z 1.39 b 1.23 b - 10 1.8 b 1.35 b 1.13 b - 20 3.3 a 2.94 a 2.24 a - 30 3.3 a 2.87 a 2.22 a -

Petunia 0 6.18 b 2.35 b 0.929 a - 10 6.56 ab 2.39 b 0.801 a - 20 6.26 ab 2.36 b 0.929 a - 30 7.65 a 2.79 a 0.971 a -

Impatiens 0 8.28 b 5.93 b 8.5 a 22.5 b 10 8.88 ab 6.21 ab 7.1 a 25.3 ab 20 10.81 ab 6.12 ab 7.6 a 24.7 ab 30 11.23 a 6.65 a 7.2 a 27.9 a

Z same lettering within columns indicate no significant differences between treatments (Tukey’s HSD 5% level). Dashes indicate data not taken.

51

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Fellbaum, C., Gachomo, E., Beesetty, Y., Choudhari, S., Strahan, G., Pfeffer, P., Kiers, E., Bucking, H., 2012. Carbon availability triggers fungal nitrogen uptake and transport in arbuscular mycorrhizal symbiosis. Proc. Natl. Acad. Sci. U.S.A. 109, 2666-2671. Finlay, R. and Soderstrom, B., 1992. Mycorrhizal Functioning. Chapman and Hall, New

York. Franken, P., 2012. The plant strengthening root endophyte Piriformospora indica:

potential application and biology behind. Appl. Microbiol. Biotechnol. 96, 1455-1464.

Gaur, An., Gaur, At., Adholeya, A. 2000. Growth and flowering in Petunia hybrida,

Callistephus chinensis and Impatiens balsamina inoculated with mixed AM inocula or chemical fertilizers in a soil of low P fertility. Sci. Hortic. 84, 151-162.

Govindarajulu, M., Pfeffer, P., Jin, H., Abubaker, J., Douds, D., Allen, J., 2005. Nitrogen

transfer in the arbuscular mycorrhizal symbiosis. Nature. 435, 819-823. Hairu, J., Jie, L., Jing, L., Wei, H., 2012. Forms of nitrogen uptake, translocation, and

transfer via arbuscular mycorrhizal fungi: A review. Sci. China Life Sci. 55, 474-482.

Hawkins, H., Johansen, A., George, E., 2000. Uptake and transport of organic and

inorganic nitrogen by arbuscular mycorrhizal fungi. Plant Soil. 226, 275-285. He, X., Critchyley, C., Bledsoe, C., 2003. Nitrogen transfer within and between plants

through common mycorrhizal networks.Crit. Rev. Plant Sci. 22, 531-567. Jin, H., Liu, J., Liu, J., Huang, X., 2012. Forms of nitrogen uptake, translocation, and

transfer via arbuscular mycorrhizal fungi: a review. 55, 474-82. Jin, H., Pfeffer, P., Douds, D., Piotrowski, E., Lammers, P., Shachar-Hill, Y., 2005. The

uptake, methabolism, transport and transfer of nitrogen in an arbuscular mycorrhizal symbiosis. New Phytol. 168, 687-696.

Javaid, A., 2009. Arbuscular mycorrhizal mediated nutrition in plants. J. Plant Nutr. 32,

1595-1618. Javot, H., Pumplin, N., Harrison, M., 2007. Phosphate in the arbuscular mycorrhizal

symbiosis: transport properties and regulatory roles. Plant Cell Environ. 30, 310-322.

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Jeffries, P., Gianinazzi, S., Perotto, S., Turnau, K., Barea, J., 2003. The contribution of arbuscular mycorrhizal fungi in sustainable maintenance of plant health and soil fertility. Biol. Fertil. Soils 37, 1-16.

Koegel, S., Boller, T., Lehmann, M., Wiemken, A., Courty, P., 2013. Rapid nitrogen

transfer in the Sorghum bicolor-Glomus mosseae arbuscular mycorrhizal symbiosis. Plant Signal Behav. 8, 8. E25229.

Krey, T., Caus, M., Baum, C., Ruppel, S., Eichler-Löbermann, B., 2011. Interactive

effects of plant growth-promoting rhizobacteria and organic fertilization on P nutrition of Zea mays L. and Brassica napus L. J. Plant Nutr. Soil Sci. 174, 602-613.

Kumar, M., Yadav, V., Kumar, H., Sharma, R., Singh, A., Tuteja, N., Johri, A., 2011.

Piriformospora indica enhances plant growth by transferring phosphate. Plant Signal Behav. 6, 723-725.

Mader, P., Veirheiling, H., Streitwolf-Engel, R., Boller, T., Frey, B., Christie, P., 2000.

Transport of N-15 from a soil compartment separated by a polytetrafluoroethylene membrane to plant roots via the hyphae of arbuscular mycorrhizal fungi. New Phytol. 146, 155-161.

Meena, K., Mesapogu, S., 2010. Co-inoculation of the endophytic fungus Piriformospora

indica with the phosphate-solubilizing bacterium Pseudomonas striata affects population dynamics and plant growth in chickpea. Biol. Fertil. Soils. 46, 169-174.

Miransari, M., 2011. Arbuscular mycorrhizal fungi and nitrogen uptake. Arch. Microbiol. 193, 77-81. Parniski, M., 2008. Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nature Rev. Microbiol. 6, 763-775. Pfeffer, P., Douds, D., Bécard, G., Shachar-Hill, Y., 1999. Carbon uptake and the

metabolism and transport of lipids in an arbuscular mycorrhiza. Plant Physiol. 120, 587-598.

Phillips, J., Hayman, D., 1970. Improved procedure for clearing roots and staining parasitic and vesicular–arbuscular fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158-161. Raaijmakers, J., Paulitz, T., Steinberg, C., Alabouvette, C., Moenne-Loccoz, Y., 2009. The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil. 321, 341-361.

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Read, D., Perez-Moreno, J., 2003. Mycorrhizas and nutrient cycling in ecosystems-a journey towards relevance? New Phytol. 157, 475-492. Ripley, B., Redfern, S., Dames, J. 2004. Quantification of the photosynthetic performance of phosphorus-deficient Sorgum by means of chlorophyll-a fluorescence kinetics. S. Afr. J. Sci. 100, 615-618. Rutto, K., Mizutani, F., Kadoya, K., 2002. Effect of root-zone flooding on mycorrhizal and non mycorrhizal peach (Prunus persica Batsch) seedlings. Sci. Hortic. 285- 295. Shamshiri, M., Mozafari, V., Sedaghan, E., Bagheri, V., 2011. Response of Petunia plants (Petunia hybrida cv. Mix) colonized with Glomus mosseae and Glomus intraradices to phosphorus and drought stress. J. Agr. Sci. Tech. 13, 929-942. Sherameti, I., Shahollari, B., Venus, Y., Altschmied, L., Varma, A., Olemuller, R., 2005. The endophytic fungus Piriformospora indica stimulates the expression of nitrate reductase and the starch-degrading enzyme glucan-water dikinase in tobacco and Arabidopsis roots through a homeodomain transcription factor that binds to a conserved motif in their promoters. J. Biol. Chem. 5, 26241-26247. Taiz, L. and Zeiger, E. 2006. In: Plant Physiology, fourth ed. Sinauer Associates, Massachusetts. Terry, N., Ulrich, A., 1973. Effects of phosphorus deficiency on the photosynthesis and respiration of leaves of sugar beet. Plant Physiol. 51, 43-47. Van Vuuren, D., Bouwman, A., Beusen, A. 2010. Phosphorus demand for the 1970-2100 period: A scenario analysis of resource depletion. Global Environ. Chang. 20, 428-439.

55

CHAPTER FOUR

THE POTENTIAL FOR THE FUNGAL ENDOPHYTE PIRIFORMOSPORA INDICA TO PROTECT PETUNIA FROM INFECTION BY PHYTOPHTHORA

NICOTIANAE

Abstract

Phytophthora nicotianae is a destructive pathogen in the ornamental horticulture

industry that causes root, crown, and stem rots as well as foliage blight. Previous studies

have shown that some biological control agents and products can suppress disease and

promote growth. Biological control products are commercially available formulations of

biological control agents. The first objective of this study was to evaluate the effect of

five commercial biological control products and a fungal endophyte,

Piriformospora indica, on growth of three species of Petunia (P.× hybrida, P.grandiflora

and P.multiflora) in the absence of P. nicotianae. The second objective was to determine

if five commercial biological control products or P. indica could suppress disease

development when these three species of petunia were inoculated with P. nicotianae.

Petunia × hybrida did not respond to biological control treatments in growth

enhancement or disease suppression. Experiments with Petunia grandiflora and Petunia

multiflora suggest that biological control agents can provide minimal suppression of

disease and improved plant growth but their effectiveness was dependent on treatment

and when plants were assessed. When compared to the control treatment, neither

biological control products nor agents had significant effects on flower number or root

dry weight of any species of petunia. P. grandiflora showed significantly higher shoot

dry weights from inoculation with Actinovate, Cease, RootShield Plus, and the 10%

56

colonized perlite treatment. All treatments were significantly higher in shoot dry weight

than the control on P. multiflora. The addition of P. nicotianae on all three species of

petunia ultimately killed all plants regardless of treatment and suppression only occurred

up to two days.

Introduction

Biological control products are commercially available formulations of biological

control agents. Biological control agents are alternatives for conventional chemical-

based pathogen control as well as for growth enhancement. Common biological control

agents for fungal diseases are bacterial and fungal antagonists and suppressors

(Nawrocka, 2013). Some of the common biological control agents for fungal pathogens

include bacteria, e.g., Streptomyces spp., Bacillus spp., Pseudomonas spp., and fungi,

e.g., Trichoderma spp. (Berger, 1996). Each biological control agent has multiple ways

to protect the host plant from abiotic and biotic attacks. Early immunity can be promoted

by a biological control agent’s ability to release elicitors and transmit signals such as

salicylic acid, jasmonic acid, reactive oxygen species, or other defense proteins

(Nawrocka and Malolepsza, 2013). Other modes for disease suppression are

mycoparasitism and pathogen antagonism.

Trichoderma spp. are omnipresent and can act as plant endophytes or saprotrophs.

There are many species and many strains in the genus. Even within species, isolates can

vary greatly in their ability as a biocontrol tool. Trichoderma spp., of the class

Sordariomycetes, are soil-inhabiting fungi that have been shown to increase biomass,

57

increase nutrient uptake, and suppress disease (Brotman et al., 2013; Nawrocka and

Malolepsza, 2013). Resistance to fungal pathogens may be due to direct antagonism,

mycoparisitism, production of antibiotics and cell wall degrading enzymes, competition

for key nutrients, and stimulation of plant defense mechanisms of a pathogen in soil or

directly on roots (Jayalakshmi et al., 2009). Two of the most common strains of

trichoderma that are currently available commercially are RootShield (Trichoderma

harzianum, strain T-22) and RootShield Plus (Trichoderma virens, strain G-41)

(BioWorks, Victor, NY). Trichoderma harzianum strain T-22 (RootShield) was selected

specifically for its ability to control a broad spectrum of soil-borne plant diseases in

different environments and within different crops (Martin, 2012). This strain was created

in a lab through protoplast fusion yielding a hybrid that serves as a biofungicide.

RootShield PLUS contains two active ingredients: Trichoderma harzianum strain T-22

and Trichoderma virens strain G-41. RootShield PLUS claims to control a broader range

of fungal pathogens including the ones managed by the original RootShield. RootShield

is the most popular and widely used biofungicide in the greenhouse industry (Haggag,

2002).

Streptomyces spp. (trade name: Actinovate), of the order Actinomycetales, is a

ubiquitous soil-dwelling actinobacterium that is capable of producing an array of

chemically diverse secondary metabolites useful for biological control (Strap, 2011). It is

considered a plant-growth-promoting rhizobacterium (PGRP) that can colonize plant

roots as well as the rhizosphere for protection from pathogens. Its hardy desiccant-

resistant spores produce antibiotics that serve multiple functions in the rhizosphere.

58

Streptomyces spp. are able to excrete antibiotics or cell-wall degrading enzymes while

also avoiding direct contact for control of the pathogen (Palaniyandi et al., 2013). In

addition, these bacteria can suppress fungal infections through competition as well as

release chitin binding proteins. Streptomyces spp. have the ability to remove nutrients,

mainly iron, from the soil which can limit the growth of pathogens.

Bacillus subtilis is a beneficial rhizobacterium that protects plant roots from

pathogen invasion. B. subtilis is able to form biofilms on plant roots where they feed on

plant exudates, produce antibiotics, compete for colonization and nutrients, and activation

of the plants defense system (Kinsella et al., 2009; Nagórska et al., 2007). The

competition of nutrients from surrounding microbes provides increased availability to the

plant potentially increasing biomass and production. Cease (Bacillus subtilis, strain QST

713) and Companion (Bacillus subtilis, strain GB03) are both commercially available

rhizobacteria.

Other fungal symbionts, such as arbuscular mycorrhizae (AM) and

Piriformospora indica, have demonstrated potential to suppress pathogen infection.

P. indica was originally isolated from the Thar Desert in India and can increase nutrient

uptake (Gosal et al., 2010) disease resistance (Deshmukh and Kogel, 2007), and biomass

production (Baldi et al, 2010) of the host plant. It is classified as a special root endophyte,

which resides only in the roots whereas endophytic fungi are able grow throughout the

plant (Yang et al., 2013). Though technically an endophyte, P. indica is commonly

referred to as an AM-like plant symbiont. Unlike AM fungi, P. indica is able to be

grown without a plant host in aseptically in an artificial media which is advantageous for

59

mass production and commercialization. P. indica also colonizes plants that arbuscular

mycorrhizae cannot, such as members of the Brassicaceae family (Deshmukh, 2006).

Phytophthora.nicotianae is an important pathogen in the horticultural industry

that causes root and stem rot and twig and leaf blight (Ferguson and Jeffers, 1999). The

pathogen is well adapted to warm temperatures and has a wide host range. Control of

P. nicotianae relies heavily on use of chemicals which increases the probability of

resistance (Joo, 2005; Olson, 2013; Sang, 2008).

This study was conducted to evaluate the potential for certain commercial

biological control agents along with an experimental biological control agent, P. indica,

to alter biomass as well as suppress P. nicotianae infection. There is currently no

published research on P. indica’s ability to suppress P. nicotianae and little research

available involving the suppression of P. nicotianae by commercial biological products.

In addition, the only published work on P. indica on floriculture crops has demonstrated

improved adventitious root formation of unrooted poinsettia and Zonal geranium cuttings

(Druege et al., 2007). Because of this lack of research we thought it important to

compare commercial biological control products to P. indica. In addition, because some

biological control products contain fertilizers or are growth promoting we investigated

their effect prior to pathogen inoculation. The objective of the first experiment was to

quantify the effect of commercial biological control products and P. indica on growth of

three species of petunia without the presence of P. nicotianae. The objective of the

second experiment was to determine the effect of commercial biological control products

and P. indica on the disease suppression of P. nicotianae on three petunia species.

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Materials & Methods

Plant materials

Petunia × hybrida ‘Potunia Plus Purple’ (Dummen USA, Hilliard, OH)

unrooted cuttings were delivered via air freight by two-day delivery. Petunia grandiflora

‘Blue Morn’ (PanAmerican Seed, West Chicago, IL) plugs grown by Rakers Acres

(Litchfield, MI) and Petunia multiflora ‘Madness Plum’(PanAmerican Seed, West

Chicago, IL) plugs grown by Green Circle Growers (Oberlin, OH) were delivered by

overnight freight in 288 plug trays.

Rooting of cuttings

Unrooted cuttings were propagated in plug trays containing 105 individual cells

(25 cm3/cell). Propagation occurred in a greenhouse using intermittent mist for 15 days.

Shade (50%) curtains were engaged when natural radiation exceeded 450 W/m2.

Heating/ventilation set points were 22/26°C and no bottom heat was used. The cuttings

received no fertilizer beyond the nutrients supplied in the propagation media (Fafard

Germination Mix, Belton, SC).

Inoculation methodology of commercial biological products

The commercial biological control products used were: Actinovate (active

ingredient = Streptomyces spp.) (Natural Industries, Inc., Houston, TX), Cease (active

ingredient = Bacillus subtilis, strain QST 713) (BioWorks, Victor, NY), Companion

(active ingredient = Bacillus subtilis, strain GB03) (Growth Products, White Plains, NY),

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RootShield WP (active ingredient = Trichoderma harzianum, strain T22), and RootShield

Plus WP (active ingredients = Trichoderma harzianum, strain T-22, and Trichoderma

virens, strain G-41) (BioWorks, Victor, NY). Each product was applied as a drench at a

rate of 177 mL/pot. Actinovate was mixed at a rate of 530 mg/L of water and reapplied

every 7 days. Cease was mixed at a rate of 14.7 mL/L of water for the first treatment and

9.7 mL/L for each subsequent treatment and was reapplied every 21 days. RootShield

WP was mixed at a rate of 298 mg/L of water and applied only at transplant. RootShield

Plus WP was mixed at a rate of 440 mg/L of water and applied only at transplant.

Companion was mixed at a rate of 2.5 mL/L of water and reapplied every 14 days.

P. indica inoculum preparation was identical to chapter 2 (see inoculation methodology

section).

Inoculation methodology of Phytophthora nicotianae

P. nicotianae isolates were provided and cultured by Dr. Steven Jeffers at

Clemson University. Four isolates of P. nicotianae (SC.00-0986, SC.01-1360, SC.02-

1076, and SC.06-0639) were grown on plates of PARPH-V8 medium at 25°C and

transferred to 10% clarified V8 agar (cV8A) (Ferguson and Jeffers, 1999). V8 broth was

prepared by mixing 100 ml of V8 Juice (Campbell Soup Company, Camden, NJ) + 1.0 g

CaCO3 + 900 ml distilled water. Fine‐textured, horticultural‐grade vermiculite was

sterilized in an oven for 24 h at 90°C, and then mixed with the V8 broth (1 part V8 broth:

2 parts vermiculite) and placed in 1 L Pyrex bottles. The necks and tops of the bottles

were covered with aluminum foil and autoclaved for 45 min, cooled to room temperature,

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and autoclaved once more for 45 min. When bottles cooled to room temperature,

three 5-mm plugs of an isolate were placed inside, tightened, and incubated at 25°C in the

dark for 14 days. Bottles were shaken gently every other day to promote uniform

colonization.

Inoculum was inspected for purity before application to plants. Each bottle had

approximately 0.5 g vermiculite plated out on non-amended cV8A under a laminar‐flow

hood. Growth of P. nicotianae was observed after 24 and 48 h at 25°C.

Expt. 1: The effect of commercial biological control agents and P. indica on plant growth

without the presence of P. nicotianae

Petunia plugs and rooted cuttings were transplanted into 12.7 cm diameter pots in

a commercial growing media (Fafard 3B, SunGrow, Belton, SC). The commercial

biological control agents were introduced to the growing media via drench application

after transplant. P. indica was applied into the growing media before transplant. The

P. indica experimental treatments consisted of a non-colonized media and media

inoculation with one of three rates of P. indica. All media treatments were comprised of

70% (by volume) commercial media (Fafard 3B, SunGrow, Belton, SC) mixed with an

additional 30% perlite (by volume). The perlite fraction was comprised of a mixture of

colonized and non-colonized perlite. The colonized perlite was 0, 33, 66 or 100% of the

total added perlite (by volume). These four inoculation treatments are referred to in this

manuscript by the percentage of the total media volume that was comprised of P. indica-

colonized perlite, e.g., 0, 10, 20, 30% (by volume).

63

After transplant, plants were kept in a greenhouse controlled by a climate control

system (Argus Controls Environmental Systems, B.C., Canada) and fertilized in each

irrigation with a 200 ppm nitrogen solution using Peters Excel 15-5-15 Cal-Mag (The

Scotts Company, Marysville, OH). Petunias were given 24 days to acclimate to the

greenhouse and associate with their designated symbiont. At the end of the experiment,

each plant was harvested for dry root and shoot weight. Leaf and soil samples were

analyzed for nutrient content.

Petunia roots were sampled to determine P. indica root colonization by the

presence of chlamydospores. Roots were first submerged with 10% w/v KOH for 3 h in

a water bath at 60°C to remove any pigmentation. Roots were then rinsed twice with

deionized water and stained with Trypan Blue for 24 h. Stain was prepared by mixing

equal volumes of water, glycerin, and lactic acid to 0.05% Trypan Blue. Roots were

viewed by mounting in 50% glycerin (Phillips, 1970).

Plants were completely randomized and at a 15.2 cm spacing. There were nine

treatments and 10 plants in each treatment. The experiment was conducted twice in

independent trials. Data were analyzed using JMP ver. 10 (SAS Institute Inc., Cary, NC).

Expt. 2: Comparison of commercial fungicidal biological products and P. indica to

manage disease caused by to P. nicotianae

Petunia rooted cuttings were transplanted into 12.7 cm diameter pots.

Commercial biological fungicides and P. indica was applied identical to Expt. 1. After

transplant, plants were kept in a greenhouse controlled by a climate control system

64

(Argus Controls Environmental Systems, B.C., Canada) and fertilized in each irrigation

with a 200 ppm nitrogen solution using Peters Excel 15-5-15 Cal-Mag (The Scotts

Company, Marysville, OH). Petunias were given 21 days (trial 1) and 31 days (trial 2) to

acclimate to the greenhouse and associate with their designated symbiont. P. nicotianae

isolates were then mixed and 2.5 mL was applied to each pot by sprinkling the colonized

vermiculite evenly around the top of the soilless media. Additional media was then

sprinkled over the top of the inoculum in order to increase moisture and protect the

inoculum from ultraviolent radiation.

Evaluations of disease severity was conducted every two days after inoculation

(DAI) using a 0 to 3 scale based on percentage of the foliage that exhibited symptoms:

0=0%, 1=1-45%, 2=46-69%, 3=70-100%. Each range of degree severity was averaged

for statistical analysis. At the end of the experiment, the roots of one plant per treatment

for Petunia × hybrida ‘Potunia Plus Purple’ was used to plate out on selective medium

(PAR(PH)) for identification of P. nicotianae. Data were analyzed using JMP ver. 10

(SAS Institute Inc., Cary, NC).

Results

Results varied for growth benefits between treatments and cultivars. A two-way

analysis of variance (ANOVA) was used to evaluate the main effects of cultivar and

treatment and the two-way interactions among factors for flower number (Table 1). The

two-way ANOVA showed no treatment by cultivar interaction while both main effects of

cultivar and treatment were significant. Regardless of treatment, P. multiflora had

65

significantly less flowers than P. grandiflora and P. × hybrida. Combined cultivars had

no significant differences between treatments when compared to the control, although P.

indica 30% colonized perlite produced significantly less flowers when compared to P.

indica 10% colonized perlite, Cease, Companion, and Actinovate.

A two-way analysis of variance (ANOVA) was used to evaluate the main effects

of cultivar and treatment and the two-way interactions among factors for shoot dry

weight (Table 2). The two-way ANOVA showed a significant treatment by cultivar

interaction and significant main effects. P. grandiflora showed significantly higher shoot

dry weights from inoculation with Actinovate, Cease, RootShield Plus, and the 10%

colonized perlite treatment. The P. multiflora control had the lowest shoot dry weight of

1.5 g, whereas Companion had the highest weight of 4.7 g (Table 2). All treatments had

significantly higher shoot dry weights versus the control. In addition, the higher

concentration P. indica (20 and 30 % colonized perlite) treatments had significantly

higher shoot dry weights than all other treatments except for companion in which there

was no difference. P. × hybrida shoot dry weight was not significantly affected by any

treatment when compared to× the control.

A two-way analysis of variance (ANOVA) was used to evaluate the main effects

of cultivar and treatment and the two-way interactions among factors for root dry weight

(Table 3). The two-way ANOVA showed no treatment by cultivar interaction and no

treatment effect yet a significant main effect of cultivar. Because of this only the main

effect of cultivar was evaluated. Thus, regardless of treatment, P. multiflora had

66

significantly less root dry weight than P. grandiflora and P. × hybrida. P. grandiflora

had the highest root dry weight of 1.7 g.

The addition of P. nicotianae on all three species of petunia ultimately killed all

plants regardless of treatment. P. nicotianae disease progress was not slowed by any

commercial fungicide when applied to P. grandiflora (Figure 4a). The 30% colonized

perlite treatment only significantly reduced disease progress at day 2 after inoculation

when compared to the control.

Commercial biological control agents and P. indica slowed P. nicotianae disease

progression only at day 6 through 8 for P. multiflora (Figure 4b). At day 6, Actinovate,

RootShield Plus, and the 20% and 30% colonized perlite treatments had significantly less

disease progression than the control. At day 8, only 30% colonized perlite reduced

disease severity. At day 10, RootShield Plus and 10% colonized perlite were the only

biological control agents to slow down P. nicotianae progression.

Biological control agents had no treatment effect on disease suppression of

P. nicotianae on P. × hybrida when compared to the control (Figure 4c). Although, at

day 12 and 14 the 10 and 20% colonized perlite treatments had significantly less disease

than the Actinovate treatment. All plant species and treatments inoculated with P. indica

were confirmed for inoculation by viewing subsets of root samples at the end of the

experiment.

67

Discussion

Results varied when growth parameters were measured on petunia species without

pathogen inoculation. Neither biological control products nor P. indica increased flower

numbers on petunia when compared with the control (Table 1). P. grandiflora showed

significantly higher shoot dry weights from inoculation with Actinovate, Cease,

RootShield Plus, and the 10% colonized perlite treatment (Table 2). All treatments had

significantly higher shoot dry weights versus the control on P. multiflora. The higher

concentration P. indica (20 and 30 % colonized perlite) treatments had significantly

higher shoot dry weights than all other treatments except for Companion in which there

was no difference. P. × hybrida shoot dry weight was not significantly affected by any

treatment when compared to the control. None of the treatments had statistically

significant effects on root dry weight on any petunia species (Table 3). In addition, when

petunia species were inoculated with phytophthora, little disease suppression was

observed by any biological control agent or product.

Although some biological control agents have the ability to increase biomass,

some can also negatively affect growth such as when the plant expends energy to activate

defense mechanisms (Mastouri, 2010). Growth promotion caused by P. indica has been

observed with many species of plants including maize, bacopa, nicotiana, artemisia, and

populus (Varma et al., 1999). In addition, flower number was increased on Coleus

forskohlii (Das et al., 2012). In the current study, the addition of P. indica did not

increase flower number in any petunia species when compared to the control, although

the P. indica 10% colonized perlite treatment was significantly higher than the P. indica

68

30% inoculated perlite treatment suggesting possible benefit from lower inoculation

concentrations. Petunia shoot dry weight was increased for some species and inoculation

concentrations. For example, the 10% colonized perlite treatment for P. grandiflora

yielded the highest shoot dry weight of all treatments and cultivars yet P. × hybrida

showed no response to P. indica inoculation.

P. indica has been demonstrated as a biological control agent. Deshmukh (2006)

presented that P. indica successfully reduced root rot on barley caused by Fusarium

graminearum. F. graminearum quantification using polymerase chain reaction revealed

a correlation between reduced root rot symptoms and the relative amount of fungal DNA

present. Additionally, research revealed that P. indica reduced the severity of wheat

pathogens that affect leaves (Blumeria graminis f. sp tritici), stems (Pseudocercosporella

herpotrichoides), and roots (Fusarium culmorum) (Serfling et al., 2006). It has also been

shown that aerial diseases can also be suppressed by root colonization with P. indica.

Colonized arabidopsis seedlings showed resistance to Alternaria brassicae infections

when compared to the non-colonized control (Nongbri et al., 2012). In addition,

F. graminearum necrotization of root tissues of barley was reduced when applied to

plants colonized with P. indica (Harrach, 2013). P. indica increased barley’s antioxidant

capacity and a correlation was found between root rot symptoms and fungal DNA.

Another study showed that P. indica has the ability to slow down reactive oxygen species

production during stress and possibly more efficiently scavenge (Baltruschat et al., 2008).

Although P. indica did not consistently reduce P. nicotianae disease pressure overtime on

any species, it did suppress disease more days in total than the commercial biological

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control agents (Figure 1). P. nicotianae disease suppression on P. grandiflora was

significantly slowed at day 2 after inoculation by the 30% colonized perlite treatment

(Figure 1a). P. indica also slowed down disease progression at days 6-10 on

P. multiflora (Figure 1b).

Trichoderma has been shown to increase biomass in plants such as in malus and

Solanum lycopersicum, whereas there was no promotion of biomass when Eruca

vesicaria was colonized with T. harzianum strain T-22 (Smith et al., 1990; Srinivasan et

al., 2009; Windham et al., 1986). Our studies only showed a significant increase in shoot

dry weight when P. grandiflora was applied with RootShield Plus and P. multiflora was

colonized with RootShield and RootShield Plus (Table 2). When tomato seeds were

pretreated with Trichoderma harzianum, strain T-22 biomass as well as total reduction of

post-germination damping off due to pythium was observed (Mastouri, 2010). In

addition, it was found that if there were no stresses, abiotic or biotic, placed on the treated

seed there was no benefit from the addition of trichoderma. In the current study only

RootShield Plus had any effect on P. nicotianae disease suppression and only on

P. multiflora (Figure 1b). It is possible only Trichoderma virens, strain G-41is beneficial

in suppressing P. nicotianae in these experimental conditions.

In addition to protection from pathogens, Streptomyces spp. has been shown to

increase plant growth in pea (Tokala et al., 2002) and increase germination of brassica

(Glick et al., 1997). The promotion of plant growth is encouraged by enhanced uptake of

iron and phosphorus as well as phytohormone production (Strap, 2011). In the current

experiment when growth parameters were measured without the addition of pathogen,

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growth was only enhanced for two species. P. grandiflora and P. multiflora had

significantly higher shoot dry weights versus the control (Table 2).

Trejo-Estrada (1998) displayed that phytophthora can be inhibited by antibiotics

produced by Streptomyces violaceusniger YCED9. Additionally, raspberry seedlings had

increased disease suppression to phytophthora from antibiotics produced by

actinomycetes (Valois et al., 1996). In the current study, Actinovate failed to suppress

P. nicotianae on all species of petunia except for P. multiflora in which was only

beneficial on day 6 after inoculation (Figure 1b).

Bacillus subtilis (Companion and Cease) also had very little effect on plant growth

and disease suppression. In the current study, Companion treated on P. multiflora

increased shoot dry weight and Cease only had significant effect on P. grandiflora by

increasing dry shoot weight (Table 2). Companion biological fungicide contains 2%

nitrogen, 3% phosphate, 2% soluble potash, 1% calcium, and 0.5% magnesium derived

from concentrated fermented plant extracts in addition to the active organism possibly

promoting the increase in shoot weight.

Cease (Bacillus subtilis, strain QST 713) is capable of producing over 30 different

lipopeptides, or antibiotics, that suppress pathogen infection (Nagórska et al., 2007).

According to Raupach (1998), Bacillus subtilis GB03 (Companion) was able to suppress

anthracnose and erwinia on cucumbers. Significant biocontrol efficacy was shown in a

study where bell peppers were colonized with Bacillus subtilis prior to infection with

Phytophthora capsici (Jiang et al., 2005). Though benefits have been previously

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documented, neither Bacillus subtilis strain suppressed P. nicotianae on any petunia

species in this study.

Conclusion

We found that the addition of biological control agents into growing media can

increase growth parameters and slow down P. nicotianae disease progression on some

species of petunia at certain days. All petunia plants colonized with P. nicotianae

reached a commercially unsalable state by one week after inoculation. Perhaps the

amount of inoculum added could have been too extreme for biological control agents to

show benefit. From the results in this experiment, none of the biological control agents

tested would be beneficial as a fungicide in a commercial setting against a P. nicotianae

infection on petunia.

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Table 1. The effects of three rates of Piriformospora indica and label rates of five biological control products on the production of flowers by Petunia grandiflora ‘Blue Morn,’ Petunia multiflora ‘Madness Plum,’ and Petunia × hybrida ‘Potunia Plus Purple’ that were grown in a greenhouse for 24 days.

Open flower number/plant z Treatment Treatment rate P. grandiflora P. multiflora P. × hybrida Treatment

main effect Control - 11.8z 5.9 11.3 9.6 bcy Actinovate 530 mg/L 15.3 6.1 13.4 11.6 a Companion 2.5 mL/L 12.1 6.8 12.9 10.6 ab Cease 14.7 mL/L 11.9 7.3 12.5 10.6 ab RootShield 298 mg/L 11.5 6.3 11.1 9.6 bc RootShield Plus 440 mg/L 13.2 6.6 11.0 10.3 abc P. indica 10% 10% 13.5 6.9 11.4 10.6 ab P. indica 20% 20% 10.1 6.4 11.1 9.2 bc P. indica 30% 30% 10.0 5.9 11.2 9.1 c Cultivar main effect 12.1 ax 6.5 b 11.7 a

2-way ANOVAw Source df P value Cultivar 2 <0.0001*** Treatment 8 0.0119* Cultivar×Treatment 16 0.2865 zData are means from two trials (n=10) at 24 DAI. yMeans that have a letter in common in the column are not significantly different based on Student’s t test with a α=0.05. x Means that have a letter in common in the row are not significantly different based on Student’s t test with a α=0.05. wTwo-way analysis of variance (ANOVA) for the main effects of cultivar and treatment; df= degrees of freedom; * and *** Represents significance at P = 0.05 and 0.001, respectively.

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Table 2. The effects of three rates of Piriformospora indica and label rates of five biological control products on shoot dry weight by Petunia grandiflora ‘Blue Morn,’ Petunia multiflora ‘Madness Plum,’ and Petunia × hybrida ‘Potunia Plus Purple’ that were grown in a greenhouse for 24 days.

Shoot dry weight (g)z Treatment Treatment rate P. grandiflora P. multiflora P. × hybrida Control - 3.6 by 1.5 d 2.6 ab Actinovate 530 mg/L 5.5 a 2.6 c 3.0 a Companion 2.5 mL/L 3.2 b 4.7 a 2.9 ab Cease 14.7 mL/L 5.3 a 2.3 c 2.7 ab RootShield 298 mg/L 3.4 b 2.7 c 2.8 ab RootShield Plus 440 mg/L 5.2 a 2.2 c 2.7 ab P. indica 10% 10% 5.7 a 2.5 c 2.4 b P. indica 20% 20% 3.4 b 3.5 b 2.4 b P. indica 30% 30% 4.0 b 4.4 a 2.4 b

2-way ANOVAx Source df P value Cultivar 2 <0.0001*** Treatment 8 <0.0001*** Cultivar×Treatment 16 <0.0001*** zData are means from two trials (n=10) at 24 DAI. yMeans that have a letter in common in columns are not significantly different based on Student’s t test with a α=0.05. xTwo-way analysis of variance (ANOVA) for the main effects of cultivar and treatment; df= degrees of freedom; *** Represents significance at P = 0.001.

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Table 3: The effects of three rates of Piriformospora indica and label rates of five biological control products on root dry weight by Petunia grandiflora ‘Blue Morn,’ Petunia multiflora ‘Madness Plum,’ and Petunia × hybrida ‘Potunia Plus Purple’ that were grown in a greenhouse for 24 days. Root dry weight (g)z

P. grandiflora P. multiflora P. × hybrida 1.7 a 0.9 c 1.3 b

Source

2-way ANOVAx df P value

Cultivar 2 <0.0001*** Treatment 8 0.2090 Cultivar×Treatment 16 0.2714 zData are means from two trials (n=10) at 24 DAI. yMeans that have a letter in common in row are not significantly different based on Student’s t test with a α=0.05. xTwo-way analysis of variance (ANOVA) for the main effects of cultivar and treatment; df= degrees of freedom; *** Represents significance at P = 0.001.

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A

B

C

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Figure 1. Disease progress for 18 days after Petunia grandiflora (A), Petunia multiflora (B), and Petunia × hybrida (C) plants were inoculated with Phytophthora nicotianae. Evaluations of disease severity were conducted every two days after inoculation (DAI) using a 0 to 3 scale based on percentage of the foliage that exhibited symptoms: 0=0%, 1=1-45%, 2=46-69%, 3=70-100%. Each range of degree severity was averaged for statistical analysis. Data were analyzed by 2-way analysis of variance, and means were separated with Fisher’s protected least significant difference (LSD) with P = 0.05. LSD values are represented by the bars above the graph.

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