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Effects of Neotyphodium uncinatum infected, loline-containing, meadow fescue–ryegrass hybrid grasses on the feeding behaviour of black beetle and red-headed pasture cockchafer larvae. 1. Assays with excised roots and potted plants

Report prepared for Cropmark Seeds Ltd

by Gary M. Barker

G. M. Barker & Research Associates

The Invertebrate Biodiversity Specialists Working in production agriculture and its interface with biodiversity conservation

______Gary M. & Maria P. Barker As Trustees for The G & M Barker Family Trust, 125 Charltons Road, Stony Creek, Victoria 3957, Australia. Tel. +61 3 5689 1236 [email protected] Report. No. 101 March 2011 2

SUMMARY Meadow fescue–ryegrass hybrids infected with Neotyphodium uncinatum endophyte were used to investigate the response of Black beetle (Heteronychus arator) and Red-headed pasture cockchafer (Adoryphorus couloni) larvae to loline alkaloids.

In one experiment, root material excised from meadow fescue-ryegrass hybrid genotypes, providing a gradient of loline concentration from 0 to 1770 µg/g DM, were feed to individual larvae in a no- choice bioassay. As controls, larvae were either offered no food (‘fasting’ treatment) or grated carrot (‘carrot’ treatment). Larval live-weight and root consumption were significantly influenced by genotypes. Over the 15 day duration of the experiment, weight gain of larvae of both species was negatively related to loline concentration in the roots. Linear regression analyses indicated 11.2% and 4.2% less weight gain per 1000 µg loline/g DM for Black beetle and Red-headed pasture cockchafer, respectively. Similarly, linear regression analyses indicated 11.3% and 2.1% reduction in root consumption per 1000 µg loline/g DM for Black beetle and Red-headed pasture cockchafer, respectively.

In the second experiment, potted plants of the same meadow fescue–ryegrass hybrid genotypes were assayed. One third of replicate plants were inoculated with 5 Black beetle larvae, another third with 5 Red-headed pasture cockchafer larvae, and a third of plants served as insect-free controls. In this experiment, the loline concentration gradient in roots ranged from 0 to 2000 µg/g DM. Live- weight changes in the larvae and larval feeding effects on plant yields were significantly influenced by genotypes. Over the 20 day duration of the experiment, weight gain of larvae of both species was negatively related to loline concentration in the roots, with 9.3% and 3.1% reduction in weight gain per 1000 µg root loline/g DM indicated by linear regression for Black beetle and Red-headed pasture cockchafer, respectively. Yield loss associated with larval infestation was indicated at 17% and 12.7% for roots, and 11% and 7.6% for foliage, per 1000 µg root loline/g DM for Black beetle and Red- headed pasture cockchafer, respectively.

The results of these experiments clearly point to the role root lolines in the feeding behaviour and growth of larvae in both Black beetle and Red-headed pasture cockchafer.

INTRODUCTION

Clavicipitaceae fungi in the tribe Balansieae form seed-borne mutualistic associations with many grasses and sedges (Bacon & White 1994, and references there in). Usually these fungi form intercellular associations with their host plant, and produce stromata on the immature infloresences or other aerial parts of the host. Species in the genus Neotyphodium Glenn, Bacon & Hanlin do not, however, produce stromata and occur as symptomless, systemic infections in the festucoid host grass (White 1988).

Many of the Neotyphodium-grass associations contain mycotoxins as secondary metabolites, some of which are known to confer resistance against herbivorous insects while some are toxic to mammals (Clay 1990, 1994; Latch 1993; Porter 1994; Rowan & Latch 1994; Bush et al. 1997; Lane et al. 2000; Clay & Schardl 2002). Of principal interest in Neotyphodium-infected grasses are the ergopeptine and ergopeptide groups of ergot alkaloids, the cyclic indole-isoprenoid lolitems, the pyrrolizidine loline alkaloids, and the pyrrolopyrazine alkaloid peramine (Bush et al. 1993, 1997; Garner et al. 1993; Rowan 1993; Porter 1994; Miles et al. 1998).

Because of their natural role in biological protection of the grass hosts, Neotyphodium endophytes are widely recognised as beneficial mycosymbionts in pastoral and turf systems. There is considerable interest internationally in the development of forage and turf grasses infected with Neotyphodium endophytes. Understanding the role of different alkaloids in protecting plants against various herbivorous pests is critical to development of endophyte-containing grasses for commercial use.

Meadow fescue (Festuca pratensis) infected with loline-producing Neotyphodium uncinatum has been shown to deter attack from several pasture insects – Argentine stem weevil Listronotus bonariensis (Patchett et al. 2008a, Jensen et al. 2009); root aphid Aploneura lentisci (Schmidt & Guy 1997); grass grub Costelytra zealandica (Popay et al. 2003; Patchett et al. 2008b). Assays with lolines topically applied or incorporated in artificial diets have been used to demonstrate the negative relationship of loline concentrations on feeding and development in a range of insects (eg. Yates et al. 1989; Patterson et al. 1991; Riedell et al. 1991; Dahlman et al. 1997; Dougherty et al. 1999; Popay & Lane 2000; Wilkinson et al. 2000; Jensen et al. 2009; Popay et al. 2009) and molluscs (Barker 2008). Of particular interest to pastoral industries in Australia and New Zealand is the adverse effect of loline alkaloids on root-feeding scarabaeid insects and the potential for development of Neotyphodium uncinatum-infected grass cultivars specifically targeting these pests. Ball et al. (1994) demonstrated that N. uncinatum infection in perennial ryegrass substantially reduced feeding by the adults of black beetle (Heteronychus arator (Fabricius)), although the alkaloid(s) involved was not determined. Edwards & Bryant (2009) and Bryan et al. (2010) demonstrated in laboratory assays that weight gains and amount of root consumption by Red-headed pasture cockchafer (Adoryphorus couloni (Burmeister)) larvae varied among N. uncinatum-infected meadow fescue genotypes and could be related to concentrations of loline alkaloids in the roots. In the same work, the response of Black beetle larvae to N. uncinatum-infected meadow fescue was inconclusive.

Black beetle is a major pest of warm temperate pastures throughout southern Australia and northern New Zealand, while Red-headed pasture cockchafer is a major pasture pest in southeastern Australia. The objective of this study was to examine the effect of variation in loline concentration in meadow fescue–ryegrass hybrids on larvae of these two scarabaeid species.

MATERIALS AND METHODS Experiments were based on clonal plant material provided by Cropmark Seeds. The plant material was sourced from a field plot trial at Morrinsville, Waikato, established to evaluate under commercial farming conditions the agronomic performance of loline-producing Neotyphodium uncinatum-infected forage grasses developed by plant breeders at Cropmark Seeds. Individual plants were shipped to Darfield, Canterbury, subdivided into clonal ramets and propagated in a gravelly sand (Osmocote amended) soil, before being shipped to Hamilton on 30 November and 6 December 2010 in polybags (no. 5). The plant material received from Cropmark Seeds comprises 50 individual plants of 8 Neotyphodium uncinatum-infected meadow fescue–ryegrass hybrids (each comprising 1 to 8 clonal lines) and 30 individual plants of an Neotyphodium-free clonal line of one of these meadow fescue–ryegrass hybrids (see Tables 1 and 2).

Experiment 1: Assay of excised roots with Black beetle and Red-headed pasture cockchafer larvae

Plant material received from Cropmark Seeds was maintained for this experiment in the gravelly sand medium at the glasshouse facilities, The University of Waikato, with regular watering and periodic trimming to ensure vigorous vegetative growth. One day before commencement of the assay, plants were removed from the potting medium and washed carefully in tap water is remove as far as practicable all soil from the roots. The plants were divided into foliage and roots components, ensuring the root material contained no meristem tissues. The harvested root material was held at 4°C in snap-lock plastic bags.

Second instar larvae of Black beetle and Red-headed pasture cockchafer were sourced from field populations at Ruakura Agricultural Research Centre, Hamilton and Purau, Banks Peninsula, respectively in early January 2011. On arrival at the laboratory, larvae were placed on the surface of potted Horotiu sandy loam soil and only those burying themselves within 30 minutes were considered healthy and subsequently used in the assay.

After a 24 hr fasting period, larvae were weighed to 1 milligram precision using electronic scales and assigned randomly to treatments. These assignments were checked to ensure mean larval weights at the start of the experiment did not differ statistically; such checks confirmed uniform dispersal of larval weight across treatments. Black beetle larvae used in the experiment ranged from 280 to 335 mg, while Red-headed pasture cockchafer larvae ranged from 180 to 211 mg live-weight.

Larvae were placed individually in 200 ml capacity plastic jars and provided with weighed quantities of root material c. 350 mg fresh weight (Figure 1). The jars were covered with a moistened paper towel and placed in a dark incubator at 20°C. Two controls were similarly set up. In one control, larvae were not provided with food (‘fasting’ treatment). In the other control, larvae were provided with grated carrot root. Each treatment was replicated 10 times, with replicates arranged in a randomised block design within the incubator.

Larvae were inspected periodically during the course of the experiment and provided with additional weighed quantities of fresh root material as required to ensure feeding ad libitum.

To enable calculation of dry weight of root provided to individual larvae, a representative subsample of root material from each treatment was weighed fresh, dried at 60°C for 48 hrs and re-weighted to determine percent moisture content. Grated carrot moisture content was similarly determined.

Figure 1. Experiment 1 – Root assay. Illustration of experimental set-up, with larvae (in this case Black beetle) in 200 ml plastic jars and provisioned with roots excised from meadow fescue–ryegrass hybrids. Jars were covered with moistened paper towels and placed in a dark incubator at 20°C for the 15 day duration of the experiment.

The experiment was terminated after 15 days. Larvae were moved to clean jars and maintained under similar conditions without food for 24 hours before being weighed to determine live-weight changes during the course of the experiment. Root and grated carrot material remaining at the end of the experiment was harvested, dried at 60°C for 48 hrs and weighted, enabling quantification of among of food material ingested during the experiment.

Subsamples of the foliage (pseudostem) and root material harvested at commencement of the experiment from each meadow fescue–ryegrass hybrid genotype were frozen at -20°C, subsequently freeze-dried and submitted to Cropmark Seeds for determination of loline concentration.

Data on changes in larval live-weight and quantities of root (and carrot) consumption were subject to analysis of variance, and related to root and pseudostem loline concentrations by regression analyses. Statistical analyses were performed using S-Plus and MS Excel.

Experiment 2: Potted plant assay with Black beetle and Red-headed pasture cockchafer larvae

Plant material received from Cropmark Seeds was maintained for this experiment individually in 5 litre plastic pots filled with Horotiu sandy loam soil at the glasshouse facilities, The University of Waikato. Plants received regular watering and periodic trimming to ensure vigorous vegetative growth. Plants were trimmed to 50 mm stubble height immediately prior to commencement of the experiment.

Second instar larvae of Black beetle and Red-headed pasture cockchafer were sourced from field populations at Ruakura Agricultural Research Centre, Hamilton and Purau, Banks Peninsula, respectively in early January 2011. On arrival at the glasshouse, larvae were placed on the surface of potted Horotiu sandy loam soil and only those burying themselves within 30 minutes were considered healthy and subsequently used in the assay.

After a 24 hr fasting period, larvae were weighed to 1 milligram precision using electronic scales and assigned randomly to treatments. These assignments were checked to ensure mean larval weights at the start of the experiment did not differ statistically; such checks confirmed uniform dispersal of larval weight across treatments. Black beetle larvae used in the experiment ranged from 286 to 318 mg, while Red-headed pasture cockchafer larvae ranged from 195 to 210 mg live-weight.

The experimental design comprised a Black beetle-infested treatment, a Red-headed pasture cockchafer-infested treatment, and an insect-free control, each replicated five times for each grass genotype. Five healthy larvae were added to each potted plant for the Black beetle-infested and Red-headed pasture cockchafer-infested treatments. One additional control was similarly set up for each insect species, where larvae were not provided with food (pots without plants – ‘fasting’ treatment).

The experiment was terminated after 20 days. The pots were destructively harvested, with surviving larvae counted, held for 24 hours at 20°C without food and then weighed to determine live-weight change during the course of the experiment. The plants were washed carefully in tap water to as far as practicable remove all soil, the roots severed from the foliage below the meristem level, and these root and foliage (pseudostem) components dried at 60°C for 48 hrs and weighted, enabling quantification of plant yields.

Subsamples of the foliage and root material harvested from each genotype at the commencement of the experiment were frozen at -20°C, subsequently freeze-dried and submitted to Cropmark Seeds for determination of loline concentration.

Figure 2. Experiment 2 – Whole plant assay. Illustration of the experimental set-up, with plants potted into Horotiu sandy loam in 5 litre pots. Within genotype, plants were grouped in threes based on visual assessment of similarity in amount of foliage, members of each triplet were then assigned randomly to the control (‘without-larval infestation’ or ‘insect-free’) and ‘with-Black beetle’ and ‘with-Red-headed pasture cockchafer’ larval infestation treatments, and finally larvae added as appropriate. The paired FhA106 plants illustrated here are from one replicate, with Black beetle larval infestation on left, and the control on the right, at day 15 of the experiment .

Data on changes in larval live-weight and plant yields were subject to analysis of variance, and related to root and pseudostem loline concentrations by regression analyses. Statistical analyses were performed using S-Plus and MS Excel.

RESULTS

Loline concentrations in plant material

Data for loline concentrations in root and pseudostem material for Experiment 1 (Root assay) and Experiment 2 (Whole plant assay) are presented in Tables 1 and 2, respectively. These data are for tissues aggregated across replicates within each genotype and experiment. With the exception of FhCF0802NIL, all plants were expected to be infected by Neotyphodium uncinatum and thus expected to contain lolines. However, for Experiment 1, FhD25 material submitted for analysis contained no detectable loline alkaloids. Similarly, for Experiment 2, no lolines were detected in FhD25 and FhA106, while in the case of FhB15, lolines were detected in pseudostem tissues only.

All parent lines from which this plant material was derived had earlier been confirmed by Cropmark Seeds as infected by N. uncinatum. It is evident from Tables 1 and 2 that either endophyte infection had not been uniformly retained during parent plant subdivision and ramet propagation, or endophyte activity and thus loline production was suppressed in some genotypes as a result of this propagation process. In presenting the results below for Experiments 1 and 2, the loline concentration data in Tables 1 and 2 are taken as representative of the loline concentrations in the plant material offered to the insects in the respective assays. It is not known to what degree loline concentrations varied between replicates within genotypes. Variation among replicates within some treatments and, in some cases, the disparity of treatment means (evident in the presented graphics) indicated that some genotypes here treated as lacking lolines may have actually included replicates in which plant material indeed contained endophyte and lolines.

Root loline concentrations ranged from 0 and 15% of those concentrations in pseudostem tissues, and across genotypes there was only a weak relationship (P > 0.05) between root and pseudostem loline concentrations (regression results not presented).

Table 1. Root and pseudostem loline concentration (µg/g DM) of meadow fescue–ryegrass hybrid genotypes used in Experiment 1 (Root assay) Roots Pseudostems NFL NAL NANL NML Total NFL NAL NANL NML Total FhCF0802NIL 0 0 0 0 0 0 0 0 0 0 FhD25 0 0 0 0 0 0 0 0 0 0 FhCF0802 633 0 0 0 633 11415 890 1468 759 14531 FhA106 908 0 0 0 908 11186 2433 2350 932 16901 FhB15 777 0 0 0 777 9502 2122 1418 623 13665 FhAB0802 970 0 164 0 1135 9396 2106 1470 737 13709 FhC2 1292 337 139 0 1768 8028 1789 1554 643 12014 FhC148 1008 0 0 0 1008 7659 1084 1608 594 10946 LpE0802 802 0 0 0 802 11992 2074 1574 1056 16697

Table 2. Root and pseudostem loline concentrations (µg/g DM) in meadow fescue–ryegrass hybrid genotypes used in Experiment 2 (Whole plant assay) Roots Pseudostems NFL NAL NANL NML Total NFL NAL NANL NML Total FhCF0802NIL 0 0 0 0 0 0 0 0 0 0 FhD25 0 0 0 0 0 0 0 0 0 0 FhCF0802 498 0 0 0 498 2657 288 382 243 3570 FhA106 0 0 0 0 0 0 0 0 0 0 FhB15 0 0 0 0 0 2843 416 451 225 3934 FhAB0802 1839 0 158 0 1997 13062 2834 1802 575 18273 FhC2 1303 0 0 0 1303 13000 2399 1784 629 17812 FhC148 865 0 0 0 865 12052 2869 1788 740 17449 LpE0802 801 0 0 0 801 12918 2690 1548 811 17967

Experiment 1: Assay of excised roots with Black beetle and Red-headed pasture cockchafer larvae

Black beetle No mortality occurred in Black beetle larvae during the experiment. Black beetle larval weight changes and root consumption of the course of the experiment were significantly influenced by the treatments (Table 3). Larvae lost weight in the ‘fasting’ treatment, but gained weight in all other treatments where either carrot or grass root material was on offer. Black beetle larval weight changes correlated positively (P < 0.001) with amount of root material consumed (Figure 3).

Table 3. Experiment 1 – Root assay. Black beetle larval initial and final live-weights, percent live- weight change, and root consumption when presented with excised roots Mean larval weight Mean root consumption Initial (mg) Final (mg) % change† (mg DM) † Fasting 308 251 f -18.3 f – Carrot 305 409 a 34.1 a 43.1 a FhCF0802NIL 306 389 b 27.3 b 33.1 b FhD25 301 343 c 14.2 c 39.2 a FhCF0802 304 332 cd 9.6 cd 31.5 bc FhA106 302 320 de 6.2 d 29.0 bc FhB15 310 330 cd 6.8 d 29.8 bc FhAB0802 311 328 cd 5.5 d 17.9 d FhC2 301 308 e 2.3 de 17.0 d FhC148 301 323 de 7.5 d 29.3 bc LpE0802 307 343 c 11.7 cd 29.8 bc

Mean 305.1 334.3 9.71 29.98

F-value df 0.818 10, 99 46.413 10, 99 37.497 10, 99 16.099 9, 90 P-value 0.612 NS <0.001 <0.001 <0.001

Fisher’s LSD0.05 16.8 6.2 5.65

†In this and subsequent tables, means within columns with different letters differ at P < 0.05 ______Gary M. & Maria P. Barker As Trustees for The G & M Barker Family Trust, 125 Charltons Road, Stony Creek, Victoria 3957, Australia. Tel. +61 3 5689 1236 [email protected] Report. No. 101 March 2011 11

Differences among meadow fescue–ryegrass hybrid genotypes in final larval weight (P < 0.001), larval weight gain (P < 0.001) and amount of root consumed (P < 0.001) were significantly negatively correlated with total loline concentration in the root material (Figures 4-6). The amount of root consumed was significantly lower for FhAB0802 and FhC2 than for other genotypes (Table 3), suggestive of a possible loline threshold effect. The negative relationship of larval weight change with total loline concentration in pseudostem material reached statistically significance (P < 0.05), (regression results not presented). The negative relationship of root consumption with total loline concentration in pseudostem material was not statistically significant (P > 0.05) (regression results not presented).

40 y = 1.0613x - 19.212 30 R² = 0.8711

0 0 10 20 30 40 50

-10 Larval weightLarval change (%) -20

-30 Root consumption (mg DM)

Figure 3. Experiment 1 – Root assay. Relationship between amount of root consumption by Black beetle larvae and their gain in live-weight. Regression line fitted to all treatments inclusive of ‘carrot’ and ‘fasting’ treatments(▲, upper right and lower left, respectively).

450 430 y = -0.0339x + 361.66 410 ► carrot R² = 0.6621 390 370 350 330 310

Larval final final Larval weight (mg) 290 270 ▲ fasting 250 0 500 1000 1500 2000

Root total loline concentration (µg/g DM)

Figure 4. Experiment 1 – Root assay. Relationship between root loline concentration and final Black beetle larval live-weights. Regression line fitted only for meadow fescue–ryegrass hybrid genotypes (i.e. without inclusion of the ‘fasting’ and ‘carrot’ treatments).

40 35 y = -0.0112x + 18.861 ► carrot 30 R² = 0.6993 25 20 15 10 5 0

-5 0 500 1000 1500 2000 Larval weight Larval change (%) -10 - 15 ▲ fasting -20 Root total loline concentration (µg/g DM)

Figure 5. Experiment 1 – Root assay. Relationship between root loline concentration and percent change in Black beetle larval live-weights. Regression line fitted only for meadow fescue–ryegrass hybrid genotypes (i.e. without inclusion of the ‘fasting’ and ‘carrot’ treatments).

45 ► carrot 40 y = -0.0113x + 37.347 35 R² = 0.7804 30 25 20 15 10

Root consumption Root (mg DM) 5 0 0 500 1000 1500 2000

Root total loline concentration (µg/g DM)

Figure 6. Experiment 1 – Root assay. Relationship between root loline concentration and root consumption by Black beetle larvae. Regression line fitted only from meadow fescue–ryegrass hybrid genotypes (i.e. without inclusion of the ‘carrot’ treatment).

Red-headed pasture cockchafer No mortality occurred in Red-headed pasture cockchafer larvae during the experiment. Larval weight changes and root consumption of the course of the experiment were significantly influenced by the treatments (Table 4). Larvae gained weight only marginally in the ‘fasting’ treatment, but gained weight substantially in all other treatments where either carrot or grass root material was on offer. Weight gain was higher on carrot and on FhCF0802NIL root material that all other meadow fescue– ryegrass hybrid genotypes. However, larval weight gain correlated poorly with amount of root DM consumed (R2 = 0.1264, P > 0.05) (graphic not presented). ______Gary M. & Maria P. Barker As Trustees for The G & M Barker Family Trust, 125 Charltons Road, Stony Creek, Victoria 3957, Australia. Tel. +61 3 5689 1236 [email protected] Report. No. 101 March 2011 13

Differences among meadow fescue–ryegrass hybrid genotypes in final larval weight (P < 0.001) and larval weight gain (P < 0.001) were significantly negatively correlated with total loline concentration in the root material (Figures 7 & 8). The relationship between amount of root consumed and total loline concentration in the root material (Figure 9) was similarly negative and statistically significant (P < 0.05). The negative relationships of weight change and root consumption with total loline concentration in pseudostem material were statistically significant (P < 0.05), but these relationships were not compelling due to the cluster dispersion of data points (graphics not presented).

Table 4. Experiment 1 – Root assay. Red-headed pasture cockchafer larval initial and final live- weights, percent live-weight change, and root consumption when presented with excised roots Mean larval weight Mean root consumption Initial (mg) Final (mg) % change (mg DM) Fasting 201 204 d 1.9 d – Carrot 196 234 a 19.4 a 15.4 b FhCF0802NIL 195 234 a 19.7 a 21.7 a FhD25 197 230 ab 16.6 b 18.8 ab FhCF0802 199 228 ab 14.7 b 18.7 ab FhA106 200 227 ab 13.8 b 18.5 b FhB15 200 231 ab 15.3 b 17.9 b FhAB0802 199 227 ab 14.2 b 17.0 b FhC2 199 220 bc 10.6 c 17.1 b FhC148 199 224 b 12.9 b 16.9 b LpE0802 199 227 ab 14.1 b 16.7 b

Mean 198.5 226.0 13.93 17.87

F-value df 0.585 10, 99 7.34410, 99 16.919 10, 99 2.388 9, 90 P-value 0.823 NS <0.001 <0.001 0.018

Fisher’s LSD0.05 8.4 3.2 3.11

240

235 y = -0.0063x + 232.47 ► R² = 0.7761 230 225 carrot 220 215

210 Larval final final Larval weight (mg) 205 ▲ fasting 200 0 500 1000 1500 2000

Root total loline concentration (µg/g DM)

Figure 7. Experiment 1 – Root assay. Relationship between root loline concentration and final Red- headed pasture cockchafer larval live-weights. Regression line fitted only for meadow fescue– ryegrass hybrid genotypes (i.e. without inclusion of the ‘fasting’ and ‘carrot’ treatments). ______Gary M. & Maria P. Barker As Trustees for The G & M Barker Family Trust, 125 Charltons Road, Stony Creek, Victoria 3957, Australia. Tel. +61 3 5689 1236 [email protected] Report. No. 101 March 2011 14

25 y = -0.0042x + 17.95 20 R² = 0.849 ► carrot

5 Larval weightLarval change (%) ▲ fasting 0 0 500 1000 1500 2000

Root total loline concentration (µg/g DM)

Figure 8. Experiment 1 – Root assay. Relationship between root loline concentration and percent change in Red-headed pasture cockchafer larval live-weights. Regression line fitted only for meadow fescue–ryegrass hybrid genotypes (i.e. without inclusion of the ‘fasting’ and ‘carrot’ treatments).

25 y = -0.0021x + 19.801 R² = 0.5517 20

15 ► carrot

5 Root DM DM Root consumption (mg DM) 0 0 500 1000 1500 2000

Root total loline concentration (µg/g DM)

Figure 9. Experiment 1 – Root assay. Relationship between root loline concentration and root consumption by Red-headed pasture cockchafer larvae. Regression line fitted only from meadow fescue–ryegrass hybrid genotypes (i.e. without inclusion of the ‘carrot’ treatment).

Experiment 2: Potted plant assay with Black beetle and Red-headed pasture cockchafer larvae

Black beetle Black beetle survival was 100%, except in FhA106 and LpE0802 where survival was 96% due to one larval death in each. Because of the generally high survival rate, these deaths were attributed to handling effect and not to treatments. Accordingly, for the replicates concerned, mean final live- weights were based on an average of four rather than five larvae.

Black beetle larval weight changes in the course of the experiment were significantly influenced by the treatments (Table 5). Larvae lost weight in the ‘fasting’ treatment, but gained weight in all other treatments where meadow fescue–ryegrass hybrid root material was on offer.

Differences among meadow fescue–ryegrass hybrid genotypes in final larval weight (P <0.001) and larval weight change (P < 0.001) were significantly negatively correlated with total loline concentration in the root material (Figures 10 & 11). The relationships with total loline concentration in pseudostem material reached statistical significance (P < 0.05), but these relationships were not compelling due to the cluster dispersion of data points (graphics not presented).

Table 5. Experiment 2 – Whole plant assay. Black beetle larval initial and final live-weights, and percent live-weight change, when presented with plotted plant material. Data are the means for the five larvae in each pot. Mean larval weight Initial (mg) Final (mg) % change Fasting 302 286 e -5.1 e FhCF0802NIL 303 407 a 34.5 a FhD25 301 379 b 25.9 bc FhCF0802 305 411 a 34.8 a FhA106 303 396 ab 30.7 ab FhB15 303 401 a 32.4 a FhAB0802 303 339 d 12.0 d FhC2 303 364 c 20.4 c FhC148 305 372 bc 22.2 c LpE0802 305 380 b 24.3 b

Mean 303.1 373.6 23.20

F-value df 0.506 9, 49 4.863 9, 49 4.840 9, 49 P-value 0.877 NS < 0.001 < 0.001

Fisher’s LSD0.05 24.4 7.94

Weight change in larvae exhibited as positive curvilinear relationship (P < 0.01) with root yield differences between pots with- and without-larval infestation (Figure 12).

Root and foliage yields varied among meadow fescue–ryegrass hybrid genotypes and were influence by Black beetle larval infestation (Table 6, Figures 13 & 14). The genotype x larval infestation interaction was statistically significant indicating genotypes responded differently to presence of larvae, or larvae were able to reduce root mass more in some genotypes.

410 y = -0.0277x + 400.1 390 R² = 0.7214 370

350

330

310 Larval final final Larval weight (mg) 290 ▲ fasting 270 0 500 1000 1500 2000 2500

Root total loline concentration (µg/g DM)

Figure 10. Experiment 2 – Whole plant assay. Relationship between root loline concentration and final Black beetle larval live-weights. Regression line fitted only for meadow fescue–ryegrass hybrid genotypes (i.e. without inclusion of the ‘fasting’ treatment).

40 y = -0.0093x + 32.011 35 R² = 0.7654 30 25 20 15 10 5

Larval weightLarval change (%) 0 - 5 ▲0 fasting 500 1000 1500 2000 2500 -10 Root total loline concentration (µg/g DM)

Figure 11. Experiment 2 – Whole plant assay. Relationship between root loline concentration and percent change in Black beetle larval live-weights. Regression line fitted only for meadow fescue– ryegrass hybrid genotypes (i.e. without inclusion of the ‘fasting’ treatment).

Differences among meadow fescue–ryegrass hybrid genotypes in root and foliage yield in the presence/absence of Black beetle larvae were significantly negatively correlated with total loline concentration in the root material (P < 0.01 and <0.05, respectively) (Figures 15 & 16). Root yield differences between with- and without-larvae were significantly lower for FhAB0802 and FhC2, indicating a possible loline threshold effect. A similar trend was apparent for foliage yields. The relationships with total loline concentration in pseudostem material with foliage yield reached statistical significance (P < 0.05), but these relationships were not compelling due to the cluster dispersion of data points (graphics not presented).

40 35 30 25 20 15 10 y = -0.0336x2 + 1.9846x + 1.7159

Larval weightLarval change (%) R² = 0.6654 5 0 0 10 20 30 40 50

Root yield difference (%) between without and with larvae

Figure 12. Experiment 2 – Whole plant assay. Relationship between percent change in Black beetle larval live-weights and root yield differences between with- and without-larval infestations.

Table 6. Experiment 2 – Whole plant assay. Root and foliage yields for plotted plants without and with Black beetle larval infestation (at five larvae/pot) Root yields (g DM) Foliage yields (g DM) Without With % yield Without With % yield larvae larvae difference larvae larvae difference FhCF0802NIL 7.68 5.14 32.7 b 5.59 3.59 35.1 ab FhD25 7.86 4.80 38.5 a 5.76 3.32 40.0 a FhCF0802 7.08 5.16 26.9 c 5.10 3.71 26.3 cd FhA106 6.94 5.16 25.3 c 5.10 3.90 23.0 b FhB15 7.28 5.40 24.5 c 5.05 4.05 19.6 de FhAB0802 7.64 7.88 -3.7 e 6.22 5.84 5.8 f FhC2 8.44 8.44 -0.2 e 6.50 5.64 12.2 e FhC148 6.92 4.72 31.6 b 5.05 3.60 28.2 bc LpE0802 7.00 5.68 18.7 d 4.90 3.91 19.8 de

Mean 7.43 5.82 21.55 5.48 4.17 23.36 Genotype effect

F-value df 20.889 8, 89 8.107 8, 44 12.236 8, 89 3.652 8, 44 P-value < 0.001 < 0.001 < 0.001 0.003 Larval effect F-value df 158.812 1, 89 95.188 1, 89 P-value < 0.001 < 0.001 Genotype x larval interaction

F-value df 8.213 8, 89 2.373 8, 89 P-value < 0.001 < 0.001

Fisher’s LSD0.05 5.35 3.83 6.34 7.97

9.00 8.00 7.00 6.00 5.00 4.00 3.00 Without larvae

2.00 With larvae Root yield yield Root(g DM/pot) 1.00 0.00

Figure 13. Experiment 2 – Whole plant assay. Bar graph illustrating root yield from meadow fescue– ryegrass hybrid genotypes without and with infestation by Black beetle larvae.

3 Without larvae 2 With larvae

Foliage yield yield Foliage (g DM/pot) 1

Figure 14. Experiment 2 – Whole plant assay. Bar graph illustrating foliage yield from meadow fescue–ryegrass hybrid genotypes without and with infestation by Black beetle larvae.

A multivariate regression model with (a) root yield in the absence of larvae, (b) Black beetle larval weight gain, and (c) root total loline concentration as predictors explained 65% of the variation in root yield differences between plants without and with Black beetle larval infestation (F-value 3,8 = 5.907, P = 0.042) .

40 y = -0.017x + 32.10 R² = 0.724 30

without and with larvae with and without -10 Root yield difference (%) between (%) difference yield Root -20 0 500 1000 1500 2000 2500 Root total loline concentration (µg/g DM)

Figure 15. Experiment 2 – Whole plant assay. Relationship of root yield of meadow fescue–ryegrass hybrid genotypes in the presence of Black beetle larvae as a function of root loline concentration.

45 40 y = -0.011x + 30.45 R² = 0.601 35 30 25 20 15

10 without and with larvae with and without 5

Foliage yield difference (%) between (%) difference yield Foliage 0 0 500 1000 1500 2000 2500 Root total loline concentration (µg/g DM)

Figure 16. Experiment 2 – Whole plant assay. Relationship of foliage yield of meadow fescue– ryegrass hybrid genotypes in the presence of Black beetle larvae as a function of root loline concentration.

Red-headed pasture cockchafer Red-headed pasture cockchafer survival was 100%. Larval weight changes in the course of the experiment were significantly influenced by the treatments (Table 7). Larvae gained minimal weight in the ‘fasting’ treatment, but gained significant live-weight in all other treatments where meadow fescue–ryegrass hybrid root material was on offer.

Differences among meadow fescue–ryegrass hybrid genotypes in final larval weight (P <0.001) and % change in larval weight (P < 0.05) were significantly negatively correlated with total loline concentration in the root material (Figures 17 & 18). The relationships with total loline concentration in pseudostem material with final larval weight and weight change similarly reached statistical ______Gary M. & Maria P. Barker As Trustees for The G & M Barker Family Trust, 125 Charltons Road, Stony Creek, Victoria 3957, Australia. Tel. +61 3 5689 1236 [email protected] Report. No. 101 March 2011 20

significance (P < 0.05), but these relationships were not compelling due to the cluster dispersion of data points (graphics not presented).

Table 7. Experiment 2 – Whole plant assay. Red-headed pasture cockchafer larval initial and final live-weights, and percent live-weight change, when presented with plotted plant material. Data are the means for the five larvae in each pot. Mean larval weight Initial (mg) Final (mg) % change Fasting 202 211 f 4.2 f FhCF0802NIL 199 244 a 22.8 a FhD25 202 239 b 18.5 b FhCF0802 200 234 c 16.8 b FhA106 202 239 b 18.0 bc FhB15 201 231 cd 14.9 de FhAB0802 199 223 e 12.1 e FhC2 199 229 d 15.2 d FhC148 198 230 d 16.1 c LpE0802 202 234 c 15.9 cd

Mean 200.3 231.2 23.20

F-value df 1.503 9, 49 17.188 9, 49 11.168 9, 49 P-value 0.180 NS < 0.001 < 0.001

Fisher’s LSD0.05 3.25 2.07

250 y = -0.0077x + 238.21 R² = 0.7315 240

230

220

210

Larval final final Larval weight (mg) ▲ fasting

200 0 500 1000 1500 2000 2500

Root total loline concentration (µg/g DM)

Figure 17. Experiment 2 – Whole plant assay. Relationship between root loline concentration and final Red-headed pasture cockchafer larval live-weights. Regression line fitted only for meadow fescue–ryegrass hybrid genotypes (i.e. without inclusion of the ‘fasting’ treatment).

Root and foliage yields varied among meadow fescue–ryegrass hybrid genotypes and were influenced by infestation by Red-headed pasture cockchafer larvae (Table 8, Figures 19 & 20). The genotype x larval infestation interaction was statistically significant for root yields, indicating genotypes responded differently in root growth to presence of larvae, or larvae were able to reduce root mass more in some genotypes.

25 y = -0.0031x + 18.574 20 R² = 0.5396

5 weightLarval change (%) ▲ fasting

0 0 500 1000 1500 2000 2500

Root total loline concentration (µg/g DM)

Figure 18. Experiment 2 – Whole plant assay. Relationship between root loline concentration and change in Red-headed pasture cockchafer larval live-weights. Regression line fitted only for meadow fescue–ryegrass hybrid genotypes (i.e. without inclusion of the ‘fasting’ treatment).

Differences among meadow fescue-ryegrass hybrid genotypes in root yield in the presence of Red- headed pasture cockchafer larvae were significantly negatively correlated with total loline concentration in the root material (Figure 21). The lower yield suppression in the presence of larvae recorded for FhAB0802 and FhC2 (Table 8), and the congruence with high root lolines (Figure 21), indicates a possible loline threshold effect. The relationship of root yield difference with total loline concentration in pseudostem material were not statistically significant (P > 0.05), albeit the slopes of the regressions were negative (graphic not presented).

9 8 7 6 5 4 3 Without larvae

2 With larvae Root yield yield Root(g DM/pot) 1 0

Figure 19. Experiment 2 – Whole plant assay. Bar graph illustrating root yield from meadow fescue– ryegrass hybrid genotypes without and with infestation by Red-headed pasture cockchafer larvae.

Similar trends were observed in the foliage yield data, with a negative relationship with root loline concentration (P < 0.05) (Figure 22). ______Gary M. & Maria P. Barker As Trustees for The G & M Barker Family Trust, 125 Charltons Road, Stony Creek, Victoria 3957, Australia. Tel. +61 3 5689 1236 [email protected] Report. No. 101 March 2011 22

Table 8. Experiment 2 – Whole plant assay. Root and foliage yields for plotted plants without and with Red-headed pasture cockchafer larvae infestation (at five larvae/pot) Root yields (g DM) Foliage yields (g DM) Without With % yield Without With % yield larvae larvae difference larvae larvae difference FhCF0802NIL 7.68 5.64 25.9 b 5.59 4.16 25.17 a FhD25 7.86 5.30 32.5 a 5.76 4.49 20.38 ab FhCF0802 7.08 5.60 20.5 b 5.10 4.65 7.49 de FhA106 6.94 5.82 15.8 cd 5.10 4.50 11.57 cd FhB15 7.28 5.70 21.3 b 5.05 4.41 12.50 c FhAB0802 7.64 7.56 0.9 e 6.22 6.08 2.01 e FhC2 8.44 8.40 0.3 e 6.50 6.18 3.88 e FhC148 6.92 5.52 19.9 bc 5.05 4.26 15.88 b LpE0802 7.00 5.98 14.5 d 4.90 4.11 15.38 bc

Mean 7.43 6.17 21.55 5.48 4.76 12.70 Genotype effect

F-value df 20.573 8, 89 11.433 8, 44 12.333 8, 89 3.209 8, 44 P-value < 0.001 < 0.001 < 0.001 0.007 Larval effect

F-value df 137.876 1, 89 32.229 1, 89 P-value < 0.001 < 0.001 Genotype x larval interaction

F-value df 86.569 8, 89 1.228 8, 89 P-value < 0.001 0.296 NS

Fisher’s LSD0.05 1.32 4.49 1.37 6.00

3 Without larvae 2 With larvae

Foliage yield yield Foliage (g DM/pot) 1

Figure 20. Experiment 2 – Whole plant assay. Bar graph illustrating foliage yield from meadow fescue–ryegrass hybrid genotypes without and with infestation by Red-headed pasture cockchafer larvae. ______Gary M. & Maria P. Barker As Trustees for The G & M Barker Family Trust, 125 Charltons Road, Stony Creek, Victoria 3957, Australia. Tel. +61 3 5689 1236 [email protected] Report. No. 101 March 2011 23

Red-headed pasture cockchafer larvae exhibited a positive relationship (P < 0.05) between their % change in live-weight and root yield differences between pots with- and without-larval infestation (Figure 23).

30 y = -0.0127x + 24.534 R² = 0.7128 25

without and withoutand with larvae 5

Root yield yield Rootdifference (%) between 0 0 500 1000 1500 2000 2500 -5 Root total loline concentration (µg/g DM)

Figure 21. Experiment 2 – Whole plant assay. Relationship of root yield of meadow fescue–ryegrass hybrid genotypes in the presence of Red-headed pasture cockchafer larvae as a function of root loline concentration.

30 y = -0.0076x + 17.32 25 R² = 0.5138 20

without and withoutand wuith larvae 5

Foliage yield yield Foliage difference (%)between 0 0 500 1000 1500 2000 2500

Root total loline concentration (µg/g DM)

Figure 22. Experiment 2 – Whole plant assay. Relationship of foliage yield of meadow fescue– ryegrass hybrid genotypes in the presence of Red-headed pasture cockchafer larvae as a function of root loline concentration.

10 y = 0.1912x + 13.494 R² = 0.473

5 Larval weightLarval change (%)

0 0 5 10 15 20 25 30 35

Root yield difference (%) between without and with larvae

Figure 23. Experiment 2 – Whole plant assay. Relationship between percent change in Black beetle larval live-weights and root yield differences between pots with- and without-larval infestations.

DISCUSSION The experimental results reported here indicate lolines in roots influence the feeding behaviour and growth of second instar larvae in both Black beetle and Red-headed pasture cockchafer.

It should be noted than the deterrancy offered by lolines is not absolute. In the present experiments, feeding occurred on all N. uncinatum-infected meadow fescue–ryegrass hybrids genotypes. Albeit reduced, the level of feeding on the genotypes with the highest root loline concentrations was often sufficient to sustain larval growth rates above that of fasting larvae.

For the most part, the relationships between root loline concentration and the response variables larval weight, root consumption and plant root yield were adequately described as linear. However, in several instances the response surface may more aptly described as sigmoid, and suggestive of a loline concentration threshold effect. In Experiment 1, root consumption by Black beetle larvae was significantly reduced on FhAB0802 and FhC2 relative to the other genotypes, consistent with loline concentrations above 1000 µg/g DM only in these grasses. Similar trends were apparent in root yield differences between pots with- and without-larval infestations in Experiment 2 for both Black beetle and Red-headed pasture cockchafer. This aspect merits further research as the existence of thresholds in loline concentration would offer specific mechanism-based objectives in plant breeding programmes. Nonetheless, it is clear that N. uncinatum-infected meadow fescue-ryegrass hybrids genotypes with high root loline concentrations are desirable for maximising disruption of feeding and growth in Black beetle and Red-headed pasture cockchafer larvae.

The closer relationship between root consumption and larval weight gain for Black beetle than for Red-headed pasture cockchafer is consistent with what is known about the species’ respective biologies. While a generalist feeder, Black beetle is less able to sustain growth on soil non-root organic matter and thus requires access to root material. The delivery of deterrent or toxic compounds via the roots of sown forage grasses offer a potential control mechanism, and in this regard, N. uncinatum-infected meadow fescue-ryegrass hybrids evidently have promise. The cockchafer feeds somewhat indiscriminately on organic matter in the root zone of soil (McQuillan & Webb 1994). The variable proportion of roots in the diet, and dilution effect of non-root organic material may affect the efficacy of root-contained lolines as a feeding deterrent against Red-headed pasture cockchafer under field conditions.

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