Endophyte and Alkaloid Effects on Survival Development, and Feeding Preference

of Black Cutworms (Lepidoptera: Noctuidae)

J. Tyler Stokes, , Carl T. Redmond, Daniel G. Panaccione, Christopher L. Schardl, and Daniel A. Potter

Department of Entomology, University of Kentucky, Lexington, KY 40546-0091

Abstract:

Survival, development, and feeding preference of the black cutworm Agrotis ipsilon

(Hufnagel) were compared on perennial rye grass Lolium perenne sp. Four strands of

grass were used: wild type, infected with Lp1 hybrid endophyte (Neotyphodium lolii X

Epichloe typhina), DMAT mutant endophyte, which produced no ergot alkaloids, LPS1

mutant endophyte, which produced no ergovaline, and the control, which was uninfected

L. perenne. Feeding preferences were shown against all three endophytic grasses, when

compared to uninfected grass, for both first and fifth-instar cutworms. When the wild

type was compared to the mutant strands, there was a significant feeding preference

towards the mutant strands. Survival to seven days, and fourteen day larval weight of the

black cutworm grown on wild type grass was reduced to less than one third of cutworms

grown on the control grass. The mutant grasses showed some significant effects in

development and weight, but differences in survival of cutworms grown on these grasses

was not as apparent as the wild type. This study suggests that ergot alkaloids, in

particular ergovaline, play a key role in insect defense. Also, the success of the wild type

grass to lower survival and development of the black cutworms may lead to applications

of this grass on turf lawns, and other places such as golf courses.

Key Words: Endophyte, Agrotis Ipsilon, Lolium Perenne

Introduction:

Many cool season grasses, including perennial rye grass Lolium perenne sp. have been

know to exhibit a symbiotic relationship with alkaloid producing fungal endophytes

(Clay 1991). Endophytes, such as Neotyphodium lolii sp., which are common in

perennial rye grass produce four classes of alkaloids: Ergot, loline, indolediterpene, and

paramine (Rowan and Latch 1994; Siegel and Bush 1997; Popay and Rowan 1994). This

endophyte, and many similar ones that grow in tall fescue have been of particular interest

to many in the cattle and horse industries due to the devastating effects the alkaloids have

on their livestock (Coleman et al. 2000; Ball et al. 2003). Ergot alkaloids, in particular,

have come under the closest scrutiny, due to the widely held belief that these are the

alkaloids most largely responsible for the livestock problems (Panaccione et al. 2001;

Wang et al. 2003). The aim to remove Egrots from grasses such as tall fescue and

perennial rye grass has led to the creation of transgenic grasses containing altered levels

of alkaloids.

L. perenne isolate Lp1, containing a hybrid Neotyphodium lolii sp. x Epichloe

typhina sp. endophyte contains almost no lolines, but increased levels of paramine and

ergot alkaloids (Panaccione et al. 2001). Two mutant strands of Lp1: LPS1, and DMAT

have been created by direct and indirect methods of gene manipulation to create L.

perenne cultivars containing no detectible ergovaline (Panaccione et al. 2001). DMAT

disables the gene responsible for the first committed step to ergot alkaloid biosyntheses.

LPS1 disrupts enzyme formation required for ergovaline, therefore allowing any ergots

created after activation of the DMAT gene, but before the LPS1 gene is needed

(Panaccione 2001).
 Many studies have shown that Endophyte infected grasses provide natural

defenses against insects, such as fall armyworm, and some coleopteran species (Braman

et al. 2002, Clay 1991, Popay and Rowan 1994). Studies however, on the black

cutworm Agrotis ipsilon (Hufnagel) response to L. perenne infected with endophytes

have proved inconclusive (Williamson and Potter 1997). The black cutworm is an

important pest of many turf grasses, including golf course tees, fairways, and greens

(Potter 1998). We hope that this study will provide some insight into possible

applications to tufgrasses, such as golf courses, that use L. perenne on many of the areas

plagued by the black cutworm. In addition, this study may provide direct evidence on the

effects of specific classes of alkaloids, e.g. ergots, and specific ergots, e.g. ergovaline, on

the black cutworm.

Materials and Methods:

Feeding preference of black cutworms

Four strands of genetically engineered Perennial Rye grass were used. The grasses,

provided by Dr. Chris Schardl (University of Kentucky, Kentucky, U.S.A), were wild

type (E+), that contained a full strength strand of the hybrid fungus Neotyphodium lolii x

Epichloë typhina, DMAT mutant, containing no detectable ergots, LPS1 mutant,

containing no ergovaline, and a control (E-) strand with no endophyte in the grass. The

grasses were grown in a green house in a soil mix of three parts Pro-Mix BX to one part

sterile top soil , in 41/2" pots , under no pesticide or herbicide pressure. Fertilization was

with Peters 20-10-20 peat-lite fertilizer, approximately every fourteen to seventeen days,

depending upon the condition and needs of the plants. Watering was once daily or

as needed. The greenhouse temp was 25°C day and 23°C night with a sixteen hour day .
The caterpillars used were freshly hatched first instar black cutworms A. ipsilon. Leaf

blades cut from the basal part of the blades were cut into 1.25 inch length pieces. Width

of blades did not differ significantly between strands, Means (SE) were: Wild-type: 2.79

(0.08) mm, DMAT: 2.83 (0.10) mm, LPS1 2.75 (0.09) mm, and Control: 2.96 (0.09) mm

respectively (n = 15 blades measured for each grass. A pair wise choice test was created,

placing six blades, three blades of each strand in a pair wise combination in 90mm radius

x 20mm tall Petri dishes in a radial, or pinwheel formation, with alternating blades. The

grass blades were chosen randomly from a bag containing blades from twenty to thirty

plants of the same strand. Petri dishes contained Whatman #1 90mm filter paper bottom,

wetted with distilled water. Ten caterpillars were placed in the center of the pinwheel

formation. Petri dishes were then sealed with parafilm. All dishes were placed in an

incubator at 26°C+-1°C with 14h/10h (L/D). Ten reps of each pair wise combination. At

both twenty four and forty eight hours the dishes were removed and the damaged

assessed. The data was analyzed qualitatively by two people, independently judging

damage to each leaf blade to the nearest ten percent.

For the fifth-instar test, the same radial formation of alternating blades was used

in pair wise combinations. The container was a Styrofoam bowl with a 120mm bottom,

covered with Whatman #1 110mm filter paper. The blades were cut at a length of 4.75

cm. For this test, to keep the blades in place, as large cutworms have the ability and

tendency to move blades, the blades were pinned in place using straight pins. A

Richmond Dental standard dental wick, wetted with distilled water was placed in-

between two blades, on the filter paper, to keep the humidity levels relatively high. The

bowls were wrapped in clear plastic wrap, and incubated as in the first-instar choice test.
The bowls were checked every three hours. The cutworm was removed from each bowl

when either, two of the three blades of one of the grasses had been eaten, or twelve hours

elapsed. The bowls were placed in a cooler at 4°C until as the cutworms were removed

until the twelve hours had elapsed. The damage was analyzed independently by two

people, rating the damage to the nearest ten five percent of each blade.

No choice rearing study of Black Cutworm

A small pile, approximately 25mm in diameter, of each strand of grass was put in the

middle of separate Petri dishes. Petri dishes contained filter paper, wet with distilled

water. Ten first-instar caterpillars were then put in the middle of the Petri dishes and the

dishes were sealed with parafilm. Eight reps of each grass and cutworms were made.

The clippings were replenished every two days, and frass removed as necessary. At

seven days, all dishes were inventoried for living caterpillars, and the caterpillars were

weighed, and each caterpillar’s instar was recorded. The caterpillars were then separated

to prevent cannibalism, and a representative sample of twenty dishes (prepared exactly as

the original dishes in this rearing study) was made for each grass, with 1 caterpillar per

dish. The caterpillars used were from the original caterpillars initially used for this

rearing study. For the control grass, there were 69 survivors, 4 first-instars, 38 second-

instars, and 27 third-instars. From this group we used the percent survivors of each

instar. For this example it was 5.8% first, 55.1% second, and 39.1% third. When these

percentages are applied to a representative sample of twenty, it gives us 1 first-instar, 11

second-instars, and 8 third-instars. These same parameters were used to determine the
representative samples for all four grasses, and their cutworms. At fourteen days the

cutworms were again counted and weighed.

Analysis of ergot, and paramine alkaloids in leaf tissue

Blades were cut from the basal section of the leaf blade and mixed with blades from

twenty to thirty plants of the same strand. They were then stored at -80°C in paper bags.

The blades were freeze dried, then analyzed by Dr. Dan Panaccione’s lab for ergot

alkaloids by methods indicated in Panaccione et al. 2001.

For Paramine analysis, the same sample of blades used for ergot analysis were sent to Dr.

Chris Schardl's Lab.

Results

Feeding preference of 1st instar black cutworms

At 24 hours, all experimental grasses showed a significant preference towards the E-

grass in all pair wise test. When compared to the E-, the E+, DMAT, and LPS1 grasses

were eaten significantly less, with all alkaloid containing grasses having <5% damage at

24h. All three trends remained significant at 48h. In a pair wise comparison between E+

and LPS1, a significantly larger amount of LPS1 had been eaten at both 24h and 48h.

This was also the case when comparing DMAT to E+, with DMAT having significantly

more damage than E+. The only pair wise comparison with no significant damage was

that of LPS1 and DMAT, with only slightly more damage on LPS1 at both 24h and 48h.

(Figure 1)
Feeding preference of fifth-instar black cutworm

The fifth-instar cutworms showed the exact same trends as those of the first instars.

Like the first-instar test, there are significant differences between the E- and the other

three grasses. When compared to the E+, the DMAT and LPS1 grasses both showed less

feeding, but no statistical significance was found. As in the first-instar test, the

comparison between the two mutants, DMAT and LPS1 showed a slight preference for

LPS1, but with no significance.

No choice rearing study

The survival rate of the instars varied greatly depending on their particular grass. Larva

reared on E- grass had about an 85% chance of survival to seven days, compared with

only about 24% survival of those on the E+ grass. The mutant grasses, DMAT, and

LPS1 were very similar with 60% and 64% survival, respectfully. Survival of the

caterpillars reared between seven and fourteen days was much less significant, with the

lowest survival rate being E+ at 88.8%, and the highest being LPS1 at 100%. The mean

larval weight of the seven day rearing group was significantly different only for the E-

grass, producing caterpillars weighing much more than any of the other three strands,

which were all relatively close. At fourteen days this changes only slightly, as the E-

grass still produced by far the largest larva; however E+ produced significantly smaller

caterpillars than either DMAT, or LPS1. The mean instar of the caterpillars is almost

identical after seven days, at right around second. This changes however, between seven

and fourteen days, and at fourteen days, there is a significant difference, LPS1 produced
significantly earlier instars than E-, and E+ produced significantly earlier instars than

either E-, or DMAT. (Table 1)

Analysis of Alkaloid content in leaf blades

(Table 2)

Discussion:

Our results showed us very clear differences in both the feeding preference, and the

growth and development of the black cutworm when reared on different strands of

grasses. The most obvious result is the survival rate of the first-instars on E+ grass,

which is much lower than any of the other three grasses. This shows clear data

supporting that ergovaline and ergine, both of which are in the wild type exclusively,

have anti-biosis effects. Other than the ability to eradicate the black cutworm, the Lp1,

and mutant infected grasses also show data that could be used to implement an Integrated

Pest Management, or IPM program. The black cutworm has been shown to be most

susceptible to predation at earlier instars (Lopez 2000). As the rearing study shows, the

E+ and LPS1 grasses produced significantly lower instars at fourteen days, when

compared to the E-. Another fact that could have IPM strategy implications is the

weight, and subsequent size of larva. At both seven and fourteen days, the larva of all

three endophyte infected grasses was significantly lower in weight. This reduction in

weight and instar may presumably allow for more casualties, either from predation or

other methods, to the survivors raised on endophyte infected grass, when compared with

those feeding on uninfected grasses.

The feeding preferences showed significant, and consistent trends away from the

endophyte infected grass, if the control (E-) was available. There was also significant
preference at early instars away from the E+ grass, compared to the mutants and control

grass. This also can directly play into applications for an IPM strategy, with reservoirs of

endophyte free, or mutant endophyte grass in less vital areas, leading cutworms in that

direction.

These two findings differ from work done by Williamson 1997, which found no

significant effect by L. perenne infected with N. lolii, compared to control (endophyte

free grass) on the black cut worm. The difference may lie in the endophytes used. The

grass type used for this study, L. perenne infected with Lp1 hybrid (E+) has been found

to contain less loline, and more ergot and paramine alkaloids than the L. perenne infected

with N. lolii used in Williamson 1997, or mutant Lp1 endophyte, (DMAT, LPS1), which

has more paramine and less lolines than Williamson 1997. (Siegel and Bush 1997).

Although these findings are very promising for places such as golf courses, and

other turf lawns, the implications into other areas such as livestock are less apparent.

Problems that devastate the livestock industry are caused by the same ergots that this

study has shown to deter herbivory the most. This study however does show some

preferences, and even some significant differences in weight, survival, and instar, when

comparing the mutant LPS1 and DMAT grasses with the control. These mutant grasses

contain simpler and less ergots(LPS1), and no ergots (DMAT). The reduction in survival

and development of the black cutworm raised on mutant grasses, as shown here, when

combined with the possible reduction in toxicosis, current insect control methods, and

general increase in grass health, compared to uninfected grass, could provide substantial

advantages to the livestock industries.

Table 1. Survival and growth of black cutworms, Agrotis ipsilon, reared from egg hatch on wild-type endophytic or
endophyte-free perennial ryegrass and on mutants lacking ergovaline (LPS1) or ergovaline and simpler ergot and
clavine alkaloids (DMAT).

7 d after neonate cohorts confined with grass 1 Status of selected survivors after 14 d2
Mean no. Mean larval Mean No. Mean larval
Grass alive wt (mg) instar alive wt (mg) Mean instar

Control (E-) 8.5  0.4 a 6.8  0.3 a 2.3  0.1 a 19/20 150  11 a 5.8  0.1 a
DMAT 6.0  0.8 b 2.5  0.3 b 2.1  0.1 a 18/20 90  12 b 5.0  0.2 ab
LPS1 6.4  0.9 ab 1.9  0.3 b 1.9  0.1 a 20/20 73  11 b 4.7  0.2 bc
Wild type (E+) 2.4  1.1 c 1.5  0.4 b 1.9  0.2 a 16/18 40  12 c 3.9  0.2 c
1
Based on eight initial replicates of 10 larvae apiece; No. alive: F = 8.67; df = 3, 28; P < 0.001; Larval wt: F = 57.8;
df = 3,24; P < 0.001; Mean instar: F = 2.31; df = 3,24; P = 0.10.
2
After 7 d, 20 representative survivors from each treatment (18 for wild type; the rest had died) were individually
reared on the same grasses for another 7 d (14 d total); Larval wt: F= 15.8, df = 3,69; P , 0.001; Mean instar: Kruskal-
Wallis nonparametric ANOVA and mean comparisons based on ranks: P < 0.001
Within columns, means followed by the same letter do not significantly differ (LSD, P < 0.05)

Table 2. Alkaloid analysis. Concentrations of ergot alkaloids in leaf blade samples fed to insects. The first
set of numbers represents mean for each alkaloid in g/g dry weight plant material  standard error; in the
second set (in parentheses), mass data have been converted to mol/kg.
Endophyte ergovaline ergine 6,7-secolysergine chanoclavine

Wild type 1.24  0.12 0.12  0.02 0.77  0.08 1.43  0.19

(E+) (2.3) (0.5) (3.2) (5.6)

LPS1 n.d.a n.d. 1.70  0.14 1.71  0.20

(7.1). (6.7)

DMAT n.d. n.d. n.d. n.d.

Control (E-) n.d. n.d. n.d. n.d.

a
n.d. none detected
Fig. 1.Results of paired choice tests with first-instar black cutworms offered
combinations of E- (CON), Wild-type (WT) E+ (N. lolii  typhinum) and genetically-
modified (LPS1, DMAT) perennial ryegrasses. Each comparison based on 10 replicates.
Larva were in a Petri dish wetted with a filter paper bottom wetted with distill water, and
provided six 1.25 in-long blade sections (three of each grass) in an alternating radial
arrangement. Asterisks denote significant differences (one-tailed paired t-test, P < 0.05).

Fig. 2. Results of paired choice tests with fifth-instar black cutworms offered
combinations of E- (CON), Wild-type (WT) E+ (N. lolii  typhinum) and genetically-
modified (LPS1, DMAT) perennial ryegrasses. Each comparison based on 10 replicates.
Larvae were starved overnight, held individually in flat-bottom poly foam soup bowls,
and provided six 4.75 cm-long blade sections (three of each grass) in an alternating radial
arrangement. Asterisks denote significant differences (one-tailed paired t-test, P < 0.05).
e a t e n
g r a s s
75 75 75
Co n t r o l Co n t r o l Co n t r o l
o f

W ild Ty pe E+ L PS1 DM A T
ne n t a g e

50 50 50

25 25 25
aet re c

*
eP

*
0
* * 0
* 0
*
g r a s s

24 h 48 h 24 h 48 h 24 h 48 h

75 75 75
L PS1 DM AT DM A T
o f

W ild Ty pe E+ W ild Ty pe E+ L PS1

50 50 50
Pe r c e n t a g e

25 25 25

*
* *
0 0
* 0
24 h 48 h 24 h 48 h 24 h 48 h
 70

Per c enta ge ofgr as s eate n ( 12 h) 60

50

40

30

20
* *
10 *
0
CO N v s . W T CO N v s . LPS1 Con v s . DM AT

60
Per c enta ge ofgr as s eate n ( 12 h)

50

40

30

20

10

0
LPS1 v s W T DM AT v s . W T LPS1 v s . DM AT
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