132

CHAPTER SIX

ENDOPHYTE STEM INFECTION AS AFFECTED BY VARIOUS FUNGICIDES ONTURF-TYPE TALL FESCUE

Abstract

Endophytes are known to enhance turfgrass agronomic performance and confer

resistance to insect herbivory. Little is known regarding the effect of fungicides to

seedlings for Rhizoctonia blight or gray leaf spot will affect endophyte viability. Various

fungicides were applied to 'DaVinci' turf-type tall fescue seedlings to determine their

effects on endophyte expression. Eight fungicides, both acropetal and localized

penetrants, were applied 7 or 21 days after establishment (DAE). Ten tillers were

harvested from each plot and cross sections of each tiller were analyzed for endophyte

presence using commercial tissue print-immunoblot test kits. In 2005, fungicide

application did not significantly affect endophyte infection rates in turf-type tall fescue 7

or 21 DAE. In 2006, however, fungicide application did significantly affect endophyte

infection rates 21 DAE. In general, propiconazole applied at 2.0 kg a.i. ha"1, seven DAE

reduced endophyte infection to 43% as compared to the control (57%). However,

myclobutanil and propiconazole (applied at 1.0 and 2.0 kg a.i. ha"1) significantly reduced

endophyte infection 21 DAE as compared to the control (90%) to 40, 40 and 7%,

respectively. These results show that propiconazole applied 21 DAE had lower endophyte

infection rates than when applied seven DAE. Endophyte mortality was reduced by the

fungicides myclobutanil and propiconazole but not eliminated. Therefore to maximize

endophytic turf, turfgrass managers should minimize certain fungicides like

propiconazole or decrease application rates especially during establishment.

133

Introduction

In recent years, tall fescue (Schedonorus phoenix (Scop.) Holub; formally know

as Festuca arundinacea Schreb.) has gained popularity and become widely planted in

lawns, athletic fields, and golf course roughs throughout the cool-humid region due to the

introduction of darker green, more narrow leaved, turf-type tall fescue cultivars. More

importantly, turf-type tall fescue is tolerant of drought because of its deep root system

which may necessitate less irrigation and less fertilizer to sustain ideal turfgrass quality.

Walker et al. (2007) found that turf-type tall fescue can be fertilized at relatively low

rates of nitrogen (74-123 kg N ha"1 yr"1) and still achieve a high visual appearance with

moderate growth compared to perennial ryegrass (Lolium perenne L.) and Kentucky

bluegrass (Poapratensis L.). In addition, turf-type tall fescue, even though it's a cool-

season turfgrass species, is a popular lawn species in the Southeast (warm-humid region)

due to its' lack of winter dormancy, remaining green throughout the year (Burpee, 1995)

unlike its warm-season counterparts. Seedling turf-type tall fescue, wherever grown, is

extremely susceptible to Rhizoctonia solani Kiihn and Pyricularia grisea which causes

the turfgrass diseases Rhizoctonia blight or brown patch and gray leaf spot, respectively

(Couch 1985).

Rhizoctonia solani causes foliar necrosis of circular or irregularly shaped patches

of necrotic turf that may exceed 1 m in diameter (Burpee and Martin, 1992). Pyricularia

grisea also causes foliar necrosis (blue-gray cast) which readily infects and kills leaf

blades which can progress down to the crown resulting in plant death. Both diseases,

brown patch and gray leaf spot, can be devastating on seedling tall fescue (< 1 yr.) killing

the entire turfgrass stand within weeks of infection. Weather conditions (hot, humid and

raining) favoring pathogen activity for both brown patch and gray leaf spot extend into

the early fall. This presents a major problem because the best time to seed cool-season

turfgrass species such as tall fescue is in the early fall so the plant can become established

before winter. Integrated methods for control against these diseases include avoiding

cultural practices that would extend the dew period to reduce of leaf wetness such as

watering or irrigating in the late afternoon and early evening and excessive nitrogen

134

applications. However, the best control against brown patch and gray leaf spot especially

on seedling tall fescue requires the use of fungicide applications (Smith et al., 1989).

Tall fescue is frequently infected with the endophytic fungus Neotyphodium

coenophialum [Morgan-Jones and Gams] Glenn, Bacon, and Hanlin) formally known as

Acremonium coenophialum. Endophytes and grasses have a mutualistic, symbiotic

association in which the plant provides the fungi with water, nutrients, and structural

refuge from the host in return the endophyte provides several benefits to the plant. These

benefits include: increased tillering and root growth, resistance to drought stress

(Arachavaleta et al., 1989), protection against insect herbivory (Rowman, 1993),

nematodes (Kimmons et al., 1990), mammalian herbivores (Bacon et al., 1977), and

fungal pathogens (Gwinn and Gavin, 1992). These benefits have made the use of

endophytes in turf an attractive alternative to chemical inputs used to enhance overall

turfgrass performance (Funk et al., 1985, 1992; Sun et al., 1990; Clarke et al., 2006).

Previous research by both White and Cole (1986) and Siegel et al. (1991) showed

that Acremonium coenophialum and other endophytes of grasses were able to inhibit the

growth of several fungal pathogens in vitro. However, disease resistance in endophyte-

infected grasses is still somewhat limited. Vincelli and Powell (1991) found that when

endophyte infection levels where high 'Manhattan IF perennial ryegrass (Lolium

perenne) had less red thread {Laetisaria fuciformis (McAlp) Burdsall) than endophyte

infection levels were low. In 1992, Gwinn and Gavin found that the survival of

endophyte-infected tall fescue seedlings was greater than that of non-endophyte-infected

seedlings in soil infested with two isolates of Rhizoctonia zeae Voorhees. Burpee and

Bouton (1993), however, reported that the progress of Rhizoctonia blight was not altered

by N. coenophialum in field grown tall fescue. Bonos et al. (2005) observed that

differences in red thread severity between endophyte-infected and non-endophyte-

infected fine fescue (Festuca spp.) occurred within the same host genotype. In 2006,

Clarke et al. reported endophyte-infected Chewings, hard, blue, and strong creeping red

fescue cultivars, selection, and crosses taken from fine fescue germplasm from the United

States and Europe, exhibited endophyte-mediated suppression of dollar spot (Sclerotinia

homoeocarpa) when compared to closely related non-endophyte-infected entries.

135

Latch and Christensen (1982) reported that seed treatments of propiconazole and

prochloraz and drenches of benomyl to potted plants eliminated an unidentified

endophyte from perennial ryegrass. Several studies have shown that triadimenol,

triadimefon, bitertanol, and propiconazole were the most effective fungicides for

eradicating Neotyphodium in tall fescue seed (Williams et al., 1984; Bilotti et al., 1989;

Maddaloni et al., 1989). Leyronas et al. (2006) found that prochloraz eliminated

Neotyphodium endophytes in perennial ryegrass and tall fescue seed. Few studies,

however, have investigated the effects on endophyte infection in plant stems as a result of

fungicide applications used to control turfgrass diseases (Dernoeden et al., 1990; Hill and

Brown, 2000). Dernoeden et al. (1990) found none of the fungicides used in the field

study (benomyl, triadimefon, iprodione, chlorothalonil, and thiram) reduced the percent

of the Acremonium endophyte in 'Regal' and 'Fiesta' perennial ryegrass infected with

endophytes. Hill and Brown's (2000) greenhouse study found that weekly applications of

the systemic fungicide, propiconazole (5%), virtually eliminated the endophyte in

seedling 'Jesup' tall fescue whereas, weekly applications of contact fungicides (terrazole

and chloroneb) had no apparent adverse effects on endophyte viability.

Rhizoctonia blight and gray leaf spot are severe diseases of seedling tall fescue

and therefore, management is limited to mainly fungicide applications. Previous research

conducted on the effects of fungicides on endophyte infection, have been limited to seed

testing and mostly greenhouse studies conducted with older fungicide formulations and

older cultivars of tall fescue. Currently, there are a wider range of commercially

available/modern fungicides for brown patch and gray leaf spot control which need to be

assessed for their effect on endophyte viability in newer turf-type tall fescue cultivars.

Therefore, the objective of this multi-year, field study was to determine the effects of

various fungicides on endophyte stem infection rates in turf-type tall fescue when applied

7 and 21 days after seedling emergence (DAE).

Materials and Methods

A field experiment was conducted from August 2005 through November 2006 at

the Purdue University, W. H. Daniel Turfgrass Research and Diagnostic Center, West

136

Lafayette, Indiana on a Stark silt-loam (fine-silty mixed mesic Aerie Ochraqualfs) with a

pH of 7.4, 67 kg P ha"1, 147 kg K ha"1, and 47 g kg"1 organic matter. Prior to planting, the

entire study area (17 x 9 m) was treated with glyphosate, N-(phosphonomethyl) glycine

to eradicate the existing turf. On the day of seeding, the entire study area was verticut to

facilitate seed to soil contact. Endophytic turf-type tall fescue ('Da Vinci') was seeded

(28 Aug. 2005 & 18 Aug., 2006) using the same seed lot both years (cold storage) at 391

kg ha"1 (Lebanon Seed Co., Lebanon, IN) using a drop spreader. This cultivar was

selected based on it's previously reported high, 84%, endophyte seed infection level

(Mohr et al., 2002). After seeding the entire study area received an application of 73 kg P

ha"1 from 6-24-24 (N-P-K) and was covered with Agrofabric Pro 17 germination blanket

(American Agrifabrics, Alpharetta, GA) to conserve moisture and promote germination.

The study area was frequently irrigated approximately three times daily via an overhead

sprinkler system to promote germination and seedling establishment.

Eight fungicides, at their label rates (Table 6-1), commonly used to control

seedling diseases were applied either 7 (12 Sept., 2005 & 1 Sept., 2006) or 21 (26 Sept.,

2005 & 14 Sept., 2006) days after emergence (DAE) to determine their effects on

endophyte infection at a spray volume of 215 gal ha"1. Emergence as defined as a uniform

stand of one-leaf seedlings which occurred on 5 Sept., 2005 and 25 Aug., 2006.

Localized penetrants included: iprodione [3-(3,5-dichlorophenyl)-N-(l-methylethyl)-2,4-

di-oxo-1-imidazolidinecarboxamide], triadimefon, and pyraclostrobin [carbamic acid, (2-

[[[ 1 -(4-chlorophenyl)-1 H-pyrazol-3-yl]oxy]methyl]phenyl]methoxy-, methyl ester].

Acropetal penetrants included: myclobutanil [a-butyl-a-(chlorophenyl)-lH-1,2,4, triazole-

1-propanenitrile], thiophanate-methyl [dimethyl 4,4'-o-phenylenebis(3-thioallophanate)],

flutolanil [N-(3-[l-methylethoxy] phenyl)-2-(trifluoromethyl) benzamide],

propiconazole, and azoxystrobin [methyl (E)-2-{2-[6-(2-cyanophenoxy) pyrimidin-4-

yloxy]phenyl}-3-methoxyacrylate]. Additionally, the plots sprayed in autumn 2005,

were sprayed again one year later (365 DAE) to determine what effect these fungicide

applications would have on endophyte infection when applied to a mature turfgrass stand.

When the study was repeated in 2006, the same eight fungicides were applied but two

additional treatments (Table 6-2) were added to include twice the label rates of

137

propiconazole (2.0 kg a.i. ha"1) and azoxystrobin (0.6 kg a.i. ha"1) because they are widely

used to control both brown patch and gray leaf spot and to assess a potential spray over-

lap situation.

Endophyte Infection

Approximately six weeks after emergence, ten tillers were randomly selected and

cut at the soil surface from each plot (1.5 x 1.5 m) to determine stem endophyte infection.

Tillers were wrapped in a damp towel and placed into a cooler of ice for transport to the

lab where they were stored overnight at 4°C. the following day, two, 2 mm cross sections

of each stem, 0.3 cm above the base, were analyzed for endophyte infection using

commercial tissue print-immunoblot test kits (TPIB) (Agrinostics Ltd. Co., Watkinsville,

GA) (Gwinn et al., 1991). For additional information on these procedures refer to

Appendix A.

General Plot Maintenance

The study site was located in full-sun with no obstructions, which was conducive

to rapid drying of the canopy in the early morning hours. Plots were mowed weekly (6.35

cm) throughout the growing season and clippings were not removed. In the absence of

significant rainfall (12 mm per week), overhead irrigation was supplementally applied

every two days (approximately 5 mm) to promote growth.

Experimental Design and Statistical Analysis

Each treatment was replicated three times and plots (1.5 x 1.5 m) were arranged in

a randomized complete-block design. All data was subjected to analysis of variance

(ANOVA) using the general linear model procedure in SAS (SAS Institute, Cary, NC).

Means were separated using protected least significant differences at the 0.05 level of

probability. Additionally, each fungicide treatment was compared to the untreated control

using pair-wise comparisons according to Dunnett's test.

138

Environmental Conditions

Weather data was collected from the Purdue University Airport, West Lafayette,

IN from Aug. 2005 through Nov. 2006. Environmental conditions, precipitation

measured as rainfall and high and low air temperature, varied between the two study

periods (Figure 6-1). Rainfall totaled 246 mm and 275 mm for Aug.-Nov. period in 2005

and 2006, respectively. Average air temperatures were slightly higher during the Aug.-

Nov. period in 2005 than in 2006 averaging 16°C and 14°C, respectively.

Results and Discussion

In 2005, fungicide application did not significantly affect endophyte infection

rates in turf-type tall fescue 7, 21, or 365 DAE (Table 6-1). In general, myclobutanil,

thiophanate-methyl, and pyraclostrobin reduced endophyte infection as compared to the

control (53%) to 33, 37, and 37%, respectively when applied seven DAE. Myclobutanil,

trifloxystrobin+triadimefon, and propiconazole reduced endophyte infection as compared

to the control (47%) to 37, 33, and 37%, respectively when applied 21 DAE. One year

later (365 DAE), propiconazole reduced endophyte infection to 27% as compared to the

control (37%). Even though there was a slight reduction in endophyte infection by the

application of these fungicides (Table 6-1), the decrease was not significant for the 7,21,

or 365 DAE applications. Dernoeden et al. (1990) reported similar results in which foliar

applications of fungicides (benomyl, triadimefon, iprodione, chlorothalonil, and thiram)

did not significantly reduce Acremonium endophyte infection in either 'Regal' or 'Fiesta'

perennial ryegrass. Hill and Brown (2000) found similar results in which weekly

applications of terrazole and chloroneb did not affect endophyte infection in seedling

"Jesup" tall fescue.

In 2006, fungicide application significantly affected endophyte infection rates 21

DAE (Table 6-2). In general, endophyte infection rates were lower than the untreated

control regardless of when propiconazole was applied which is consistent with previous

research in which the fungicide was applied to plant tissue (Harvey et al., 1982; Hill et

al., 1990; Hill and Brown, 2000) or as a seed treatment (Harvey, 1982). Propiconazole

applied at 2.0 kg a.i. ha"1 seven DAE, reduced endophyte infection to 43% as compared

139

to the control (57%). However, myclobutanil and propiconazole (applied at 1.0 and 2.0

kg a.i. ha1) significantly reduced endophyte infection 21 DAE as compared to the control

(90%) to 40, 40 and 7%, respectively. Hill and Brown (2000) reported similar results in

which propiconazole (4.0 g a.i. L"1) applied seven DAE reduced endophyte infection

(9%) compared to the untreated control (83%) for 'Jesup' tall fescue. Hill and Brown

(2000) also reported that propiconazole applied 21 DAE reduced endophyte infection

(55%). This study shows that increasing the rate of propiconazole from 1.0 to 2.0 kg a.i.

ha"1 decreased endophyte viability from 40 to 7%.

This study as well as other studies (Dernoeden et a l , 1990; Hill and Brown, 2000)

found that the use of some fungicides in seedling tall fescue adversely affected endophyte

viability however, effects are somewhat variable. The greatest risk in decreasing

endophyte viability occurs when more than the recommended label rate of these

fungicides are applied or when overlapping of fungicide occurs resulting in higher

application rates then typically suggested. It is important to note that endophyte mortality

was reduced but not eliminated by these fungicides used for brown patch and gray leaf

spot control. Due to beneficial attributes of endophytic fungi in tall fescue and the

extensive use of these fungicides to control Rhizoctonia blight or gray leaf spot on

managed turfgrass areas, these findings are important to turfgrass managers enabling

them to properly care for endophyte-infected tall fescue. Therefore to maximize

endophytic turf, turfgrass managers should minimize certain fungicides like

propiconazole or decrease application rates especially during establishment.

140

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144

Table 6-1. Percent endophyte stem infection of 'Da Vinci' turf-type tall fescue after eightfungicides were applied 7 and 21 days after emergence (DAE) in 2005.

Fungicide

IprodionePyraclostrobinTrifloxystrobin+TriadimefonAzoxystrobinFlutolanilMyclobutanilPropiconazoleThiophanate-methylNone

ANOVARepFungicide

TopicalActivity!

LPLPLPAPAPAPAPAP—

ApplicationRate

kg a.i. ha'1

3.10.60.90.36.41.51.06.1—

Endophyte Infection!

7 DAE

503750606033503753

NSNS

21 DAEo/o

474330435037375047

NSNS

365DAE

333737373053274037

NSNS

f AP=Acropetal Penetrant, LP=Localized PenetrantJTen tillers from each 2.3 m2 plot were tested for endophyte stem infection on 5 Nov.,2005 using a tissue print-immunoblot assay for Neotyphodium spp.NS refers to non-significantTurf-type tall fescue ('Da Vinci') was provided by Lebanon Seed Co., Lebanon, EN andseeded at rate of 391 kg ha'on 28 Aug., 2005. Emergence as defined as a uniform standof one-leaf seedlings occurred on 5 Sept., 2005.Seven DAE was applied on 12 Sept., 2005 and 21 DAE was applied on 26 Sept., 2005 ata spray volume of 215 gal ha"1.Randomized complete block design with three replications.

145

Table 6-2. Percent endophyte stem infection of 'Da Vinci' turf-type tall fescue after eightfungicides were applied 7 and 21 days after emergence (DAE) in 2006.

Fungicides

IprodionePyraclostrobinTrifloxystrobin+TriadimefonAzoxystrobinAzoxystrobinFlutolanilMyclobutanilPropiconazolePropiconazoleThiophanate-methylNone

ANOVARepFungicide

TopicalActivityt

LPLPLPAPAPAPAPAPAPAP—

ApplicationRate

kg a.i. ha"3.10.60.90.30.66.41.51.02.06.1—

Endophyte Infection^

7 DAE

7383709087806773436757

NSNS

21 DAE__o/o

83808767838340*40*7 ***

8090

NS***

t AP=Acropetal Penetrant, LP=Localized Penetrant{Ten tillers from each 2.3 m2 plot were tested for endophyte stem infection on 26 Oct.,2006 using a tissue print-immunoblot assay for Neotyphodium spp.*, *** and NS refer to significant at the 0.05 and 0.001 level and non-significant relative to theuntreated control using pair-wise comparisons with the untreated control according to Dunnett'stest, respectively.Turf-type tall fescue ('Da Vinci') was provided by Lebanon Seed Co., Lebanon, IN andseeded at rate of 391 kg ha^on 18 Aug., 2006. Emergence as defined as a uniform standof one-leaf seedlings occurred on 25 Aug., 2006.Seven DAE was applied on 1 Sept., and 21 DAE was applied on 14 Sept., 2006 at a sprayvolume of 215 gal ha1.Randomized complete block design with three replications.

146

Table 6-3. Analysis of variance (ANOVA) for both 2005 and 2006 study periods.ANOVARepDAEFungicideDAE*Fungicide

2005NSNSNSNS

2006NSNS***

*e, *** and NS refer to significant at the 0.05 and 0.001 level and non-significant, respectively.

147

200

150

1ca:

50

RainfallHighLow

Total = 246 mm

Jk

Total = 275 mm

Aug. Sept. Oct. Nov.

2005

Aug. Sept. Oct. Nov.

2006

40

30

20 Q

10

-10

-20

(D

2CDQ.

Figure 6-1. Daily temperature (high and low) and rainfall during Aug. through Nov. of2005 and 2006 in West Lafayette, IN.