Influence of Botrytis cinerea (Helotiales: Sclerotiniaceae) infected leaves ofVitis vinifera (Vitales: Vitaceae) on the preference of Epiphyas postvittana(Lepidoptera: Tortricidae)

Syed Z M Rizvi,1 Anantanarayanan Raman,1,2,* Warwick Wheatley,1 Geoffrey Cook1 and Helen Nicol1

1School of Agricultural and Wine Sciences, Charles Sturt University, Orange, NSW 2800, Australia.2E H Graham Centre for Agricultural Innovation, Charles Sturt University, Orange, NSW 2800, Australia.

Abstract Herbivorous insects use sensory cues to choose their host plants for feeding and/or oviposition by assessinghost quality. Olfactory, contact and visual cues of the host mediate such choices. The right-host choice foroviposition by a lepidopteran is essential for the performance of its progeny. In natural conditions, plants areoften and concurrently attacked by both herbivorous insects and pathogenic fungi. In such a three-wayrelationship, the interaction between the plant and insect is usually influenced by the fungal population, andsuch an influence can be either mutualistic or antagonistic. In the present study, we tested the three-wayrelationship using the system, Epiphyas postvittana–Vitis vinifera–Botrytis cinerea. We sought answers to thequestions: (1) whether the females of E. postvittana prefer to oviposit on V. vinifera leaves infected byB. cinerea; and (2) whether the larvae of E. postvittana prefer to feed on V. vinifera leaves infected byB. cinerea. We found that the host-seeking gravid females of E. postvittana ‘tested’ the infection status of thehost plant using olfactory, visual, and tactile cues; in consequence, they laid significantly fewer eggs on themoderately (30–60%) and intensely (90–100%) infected leaves of V. vinifera. The neonate larvae preferred tofeed on mildly (5–10%) and moderately (30–60%) infected leaves, as against the uninfected (control) leaves,and showed no preference for intensely (90–100%) infected leaves. External and internal examination of thelarvae established that the larvae fed on B. cinerea-infected leaf because viable conidia of B. cinerea occurredon the body surface and within the gut of the neonate larvae.

Key words grey mould, light brown apple moth, olfactory cue, oviposition deterrence, tactile cue.

INTRODUCTION

Chemical cues mediate plant–insect interactions (Schoonhovenet al. 2005; Bruce et al. 2010). Plant-feeding insects usesensory cues such as olfactory, contact chemoreceptory andvisual to identify and assess the food, mates, predators, com-petitors and oviposition sites (Schoonhoven et al. 2005; Tasinet al. 2011). Unlike visual and contact chemoreception, olfac-tory cues are considered helpful to plant-feeding insects ineither recognising or rejecting the host plants from a distance(Bruce et al. 2005; Hallem et al. 2006). Nevertheless, the roleof olfactory cues from the host plant is not yet fully explained(Städler 2002). That the olfactory cues emitted by a plant canguide a gravid lepidopteran on the suitability of a site foroviposition and nutrition for her progeny remains unresolved.We, however, know that olfactory cues are critical for locatingan appropriate host for oviposition; once the lepidopteran is onthe target plant, the contact cues, coupled with the olfactorycues, influence the lepidopteran in assessing the plant in ena-

bling it to either progress with the oviposition or not (Fosteret al. 1997; Tasin et al. 2011). Based on the sensory cues of aplant-feeding insect, judgment on the appropriateness of a hostplant is essential for the success of performance of its progeny(Gripenberg et al. 2010). The gravid female lepidopteran usesthe olfactory and visual cues to locate and identify the hostplant, but the decision to either accept or reject the plantdepends on contact cues, which are often plant-surface com-pounds and texture (Foster et al. 1997; Mehar et al. 2006).

In the natural environment, several organisms attack plants;among these, insects and fungi are associated with a majorityof plants, establishing a three-way interacting system. In suchcontexts, the fungi influence the interaction between the insectand the plant, which can be either mutualistic or antagonistic,and, occasionally, neutral as well (Hatcher 1995; van Dam2009). Tack and Dicke (2013) have explored the ecology ofplant–microbe–insect interactions in a community context,whereas Biere and Tack (2013) have characterised the evolu-tionary consequences of plant–microbe–insect interactions.Fungi induce variations in the chemistry of the plant that canmodify the volatiles profile of the plant (Cardoza et al. 2003b;Raman et al. 2012). Such induced variations in the host-plant*araman@csu.edu.au

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volatiles could be recognised by the olfactory receptors ofLepidoptera as either an attractant (Cardoza et al. 2003b) or adeterrent (Tasin et al. 2012).

Botrytis cinerea is a necrotrophic plant pathogen that occursworldwide inducing the grey-mould disease on Vitis viniferaaffecting its leaves and berries (Fournier et al. 2013).B. cinerea commonly occurs in the Australian viticulturalregions in varying intensities (Nair et al. 1995). Fungal hyphaeactively penetrate the cell and enzymatically digest the leafcells and alter the leaf integrity and physiology (Volpin &Elad 1991; Carlile et al. 2001; Jansen et al. 2009). Infectionof V. vinifera by B. cinerea induces the biosynthesis ofbehaviour-modifying volatiles, which influence the hostchoice made by different insects infesting V. vinifera (Tasinet al. 2012). Often, B. cinerea populations co-occur with thelarvae of E. postvittana; the conidia and hyphae of B. cinereahave been found in the gut of mature larvae of E. postvittana.The larvae also facilitate the dispersal of B. cinerea by trans-mitting the spores that adhere to it and also by passing viaexcreta (Bailey et al. 1997; Lo & Murrell 2000). The feedingdamage inflicted by the larvae of E. postvittana render theberries amenable to B. cinerea infection by creating a settlingsite for and germination of the conidia (Lo & Murrell 2000).Whether the larvae derive any benefit from the fungus orwhether it is co-occurrence remains unanswered.

Epiphyas postvittana is native to Australia and has invadedNew Zealand, England, Ireland, the Netherlands, Sweden, andHawaii and California in the USA (Bradley et al. 1973;Wolschrijn & Kuchlein 2006; Suckling & Brockerhoff 2010).E. postvittana is known to feed on more than 500 plant speciesbelonging to 121 families (Suckling & Brockerhoff 2010). Inthe field, female lays 300–400 eggs in discrete batches of 4–77(Danthanarayana 1975). A female of E. postvittana can lay upto 1500 eggs, but the egg-laying capacity is strongly influ-enced by the nature of food consumed and temperature expe-rienced during its larval stages (Danthanarayana 1983). Eggsoccur partly overlapping each other, pale yellow–white andtranslucent when fresh. Neonate larvae emerge in 8–13 daysafter oviposition. By swinging on the silken thread, theyproduce from their salivary secretions; they disperse by windaction to other potential sites on the host plant. The dispersedlarvae spin a web and feed within the shelter created by theweb. Duration of larval development from egg to pupae is26.7 ± 0.4 (mean ± SE) days at 20°C (Danthanarayana 1975).Pupation occurs within the shelter. Pupae are greenish-browninitially but turn fully brown when mature. Male pupae bearfour segments and are 7.1–7.7 mm long, whereas the femalepupae bear three segments and are 9.5–9.8 mm long(Danthanarayana 1975). Adults of E. postvittana emerge in8–10 days after pupation. The lifespan of an adult male andfemale E. postvittana at 21°C is 16–44 and 31–34 days,respectively (Danthanarayana 1975).

In the present study, we tested the three-way relationshipusing the three-way interacting system: E. postvittana–V. vinifera–B. cinerea. Many experiments were conducted toaddress the following questions: (1) whether the females ofE. postvittana prefer to oviposit on V. vinifera leaves infected

by B. cinerea; and (2) whether the larvae of E. postvittanaprefer to feed on V. vinifera leaves infected by B. cinerea.Tactile stimuli indeed play a role in host selection, whereasliterature (Reddy & Raman 2011; Machial et al. 2012) rein-forces that olfactory stimuli play the principal role in host-plant selection. Using this dictum, in this study, we haveconsidered the olfactory stimuli as the principal and earliesttrigger factor. In securing the answers, we compared theovipositional preference of E. postvittana to B. cinerea-infected and uninfected (control) leaves of V. vinifera at threelevels of infection referred to as ‘mild’, ‘moderate’ and‘intense’, where the adult E. postvittana female could use thesensory cues, such as olfactory, tactile and visual. The effect ofodour of B. cinerea-infected leaves on ovipositional behaviourof E. postvittana was tested by allowing the females touse only olfactory cues. The olfactory response of maleE. postvittana was also compared with B. cinerea-infected anduninfected (control) leaves of V. vinifera. The olfactoryresponse of the larvae towards the uninfected (control) leavesand B. cinerea-infected leaves of V. vinifera was compared.The ability of larvae of E. postvittana to feed on B. cinerea-infected leaves was also examined.

MATERIAL AND METHODS

Insect culture

Eggs of E. postvittana were obtained from pure-laboratorycultures of insects maintained at the School of Agriculture,Food and Wine, Waite Campus, The University of Adelaide,Adelaide. The larvae were reared on a Phaseolus lunatus (limabean)-based semi-synthetic diet (Cunningham 2007). Plasticboxes (35 × 20 × 4 cm) were filled to about a third with thediet and E. postvittana eggs on paper rafts were placed inside.The set-up was maintained at 20–22°C, 60–80% RH, and a16 L : 8D regimen. Once the larvae pupated, the male andfemale pupae were sexed and segregated considering the totalbody length and the number of abdominal segments. On adultemergence, one female and one male were transferred to acorrugated wall Dixie cup. An aqueous-honey solution (10%)with 0.1% sorbic acid soaked in a cotton ball was provided tothe adult pair for feeding. Rafts of laid eggs were cut out fromthe Dixie cup wall and placed in semi-synthetic diet containingplastic boxes to maintain the culture. A few of the egg massrafts were also refrigerated at 4–6°C. A 24-h-old female wasisolated with a similar-aged male in the Dixie cup. After 24 h,only those females that laid about 10 eggs were used in theexperiments, the details of which are provided in the followingsections. Adult females were not exposed to either uninfectedV. vinifera (control) or B. cinerea-infected V. vinifera, prior toexperiments.

Fungus culture and preparation ofspore suspension

Mycelia of B. cinerea were isolated from the infected berriesof V. vinifera cv. Chardonnay collected from the Charles Sturt

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University vineyard (CSU), Orange, NSW in February 2013.Source material was provisionally identified based on micro-scopic observation of the mycelia and conidia (Khazaeli et al.2010). B. cinerea was later confirmed by Michael Priest (Prin-cipal Mycologist and Plant Pathologist, Department ofPrimary Industries, Orange, NSW). The determined fungalculture was maintained on potato-dextrose agar (PDA) (FisherScientific, Inc., Scoresby, Vic., Australia) at 22°C and12 L : 12D.

To prepare the spore suspension, fungal cultures with c.2-week-old conidia were flooded with 5 mL of sterile wateradded with 0.01%. Tween 80 (Acros Organics, Geel, Belgium)solution, and the surface of the B. cinerea colonies wassmeared with a bent platinum wire streaker. The density of theconidial suspension was determined with a Neubauer chamberand adjusted to 106 conidia/mL (Trigiano et al. 2008). Thissuspension was used for the inoculation of leaves ofV. vinifera.

Plant culture

Forty-nine rootlings of V. vinifera cv. Chardonnay (hereafterreferred to as V. vinifera) obtained from a registered vinenursery in Trentham Cliffs, south-western NSW, were raised innursery-grade potting mixture (Osmocote, Plus Organics Veg-etable & Herb Mix, Sydney, NSW, Australia) in plastic pots(20.91 dm3) in an insect-proof glasshouse at CSU at 25–27°Cand 16 L : 8D in October 2012. All plants were watered uni-formly every day. Neither insecticides nor fungicides wereapplied. At the start of the experiments in April 2013, theraised plants were 6 months old with several fully expandedleaves. Mature, dark-green leaves (9–12 cm long) were used inall experiments. While considering the use of excised leaves inthis experiment, we relied on Schmelz et al. (2001), who indi-cate that the process of senescence could be delayed inlaboratory-based assays, although only modestly, when appro-priate care is exercised. Nevertheless, excised leaves have beenfollowed in insect–plant interaction assays even in recenttimes, e.g. Sharma et al. (2005) and Michel et al. (2010) forreasons of experimental convenience. Peros et al. (2006) hadused excised leaves to characterise fungus–plant (Erysiphenecator, Erysiphales: Erysiphaceae–V. vinifera) interactions.Keeping the previous data in view, we used excised leavescoupled with a damp-filter paper to maintain a reasonable levelof moisture in our experiments, thus delaying senescenceduring experiments.

Infection level of B. cinerea on the leaves ofV. vinifera

Excised leaves (petioles + laminae) of V. vinifera were surfacesterilised with 1% NaClO solution for 5 min and washed withsterile water (3×). After surface sterilisation, leaves wereimmediately sprayed with the spore suspension on the sterilebench of a horizontal laminar airflow cabinet (HWS120,Clyde-Apac, Sydney, NSW, Australia). Control leaves weresprayed with sterile water. Inoculated and control leaves were

placed in a zip-lock plastic bag (35 × 40 cm) supplied with adamp filter paper and incubated at 22°C and 12 L : 12D. After4–6, 9–11 and 14–16 days, the lesions on the B. cinerea-infected leaves were evaluated as 5–10% being ‘mild’,30–60% being ‘moderate’ and 90–100% being ‘intense’,respectively. The infection level of B. cinerea-induced lesionswas measured in percentage, from the photographs made witha digital camera (D-60, Canon, Tokyo, Japan), and calculatingthe area of infection using ‘ImageJ’, an open-source imageprocessing software (Abramoff et al. 2004).

Bioassay of oviposition behaviour

Experiments were conducted using excised V. vinifera leavesbearing infection levels as mild, moderate and intense alongwith appropriate control leaves. The experiments were con-ducted in custom-made Pyrex® glass devices (details in thefollowing sections) at 20–22°C, 60–70% RH and 16 L : 8D(four units of 28W slim-line diffuse flush fluorescent lighttubes, with an additional polyethylene sheet acting as a dif-fuser attached). On completion of every experiment, thecustom-made glass devices were washed thoroughly with acommercial-detergent solution, followed by a 70% ethanolrinse and oven-dried at 110°C for 8 h before setting up the nextexperiment. A two-choice experiment was set up to evaluatethe ovipositional preference pattern of E. postvittana onB. cinerea-infected and uninfected (control) leaves. Ano-choice experiment was set up to evaluate the rate ofoviposition on B. cinerea-infected and uninfected (control)leaves using an identical but a different glass device. Details ofglass devices used in the two experiments are explained in thefollowing paragraphs.

Two-choice experiment

The two-choice experiment was conducted using a custom-made glass device, which consisted of two 1000-mL flasksconnected by a horizontal glass tube (30 cm long, 3 cm wide).The diameter of the horizontal tube was chosen in such a waythat its ends fitted snugly into the mouths of the flasks; thejunctions were sealed tightly with Parafilm®. At the mid-pointof the horizontal glass tube, a 2-cm wide circular port was cutfor the introduction of insects. Three B. cinerea-infectedleaves (5–10%) of V. vinifera (7.00 ± 0.23 g; mean ± SE) wereplaced in one flask and three control leaves of V. vinifera(7.00 ± 0.18 g; mean ± SE) were placed in the other. Fivegravid E. postvittana adults were introduced through the port,allowing the insects to make their choice of either B. cinerea-infected or control leaves (Fig. 1). The port was covered withParafilm. After 72 h, the insects, all of which remained aliveand active, were removed by disassembling the device.Choices made by the insects were recorded by counting thenumbers in either of the flasks and the number of eggs laid onB. cinerea-infected and control leaves were counted using astereo-binocular microscope (S-20, AIS Instrument Services,Croydon, Vic., Australia). The same procedure was repeatedfor leaves with 30–60% and 90–100% infection levels, eight

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times each. In each experiment, the location of infected andcontrol leaves was randomised. Each treatment was replicatedeight times by using four glass devices on each day.

No-choice experiment

This experiment was done following Yan et al. (1999) withlittle modification. Either two B. cinerea-infected leaves(5–10% or 30–60% or 90–100%) or two appropriate controlleaves of V. vinifera (4.2 ± 0.21 g; mean ± SE) were placedinside a 500-mL Pyrex glass conical flask. A gravidE. postvittana was introduced into the conical flask, and theopening was sealed immediately with Parafilm (Fig. 2). After72 h, the moths were removed, and the eggs laid on the leaveswere counted. Each treatment was replicated 10 times.

Effect of volatiles on oviposition

In this experiment, ovipositional rate of E. postvittana wastested in response to the volatile compounds emitted fromB. cinerea-infected leaves or appropriate control leaves ofV. vinifera. A 200-mL Dixie cup with vertical ridges andfurrows was used as an ovipositional device. The bottom of

each cup was removed, wrapped in plastic wrap and pierced 30times using a safety pin to allow the volatiles to pass inside thecup. This cup was then fixed over a 250-mL beaker, whichincluded a damp filter paper. Either two control leaves or twoB. cinerea-infected leaves (5–10% or 30–60% or 90–100%) ofV. vinifera (3.96 ± 0.12 g; mean ± SE) were placed inside thebeaker. The junction point of the beaker with the Dixie cupwas sealed with Parafilm (Fig. 3). A 24-h-old matedE. postvittana female was introduced into the Dixie cup, andthe cup was covered with Parafilm. After 72 h, moths wereremoved and eggs deposited on the cup counted. Each treat-ment was replicated 10 times.

Y-tube experiment

The behavioural responses of adult males E. postvittana (24–48 h old) towards B. cinerea-infected leaves (5–10% or

Fig. 1. Custom-made glass device (horizontal tube connected to two 1000-mL flasks) for the two-choice experiment of adults ofEpiphyas postvittana (not to scale). f, flask; hgt, horizontal-glass tube; il, infected leaves; m, moth; pe, port of entry; ps, parafilm seal;ul, uninfected leaves.

Fig. 2. Custom-made glass device for the no-choice experimentof adults of Epiphyas postvittana (not to scale). f, flask; il, infectedleaves; m, moth, ps, parafilm seal; ul, uninfected leaves.

Fig. 3. Custom-made device to test the effect of volatiles onoviposition behaviour of Epiphyas postvittana (not to scale). b,beaker; d, Dixie cup; il, infected leaves; m, moth; ps, parafilmseal; pw, plastic wrap; ul, uninfected leaves.

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30–60% or 90–100%) and appropriate control leaves ofV. vinifera were measured using aY-tube olfactometer (OLFM-YT-2425F;Analytical Research Systems, Gainsville, FL, USA)having a stem (2.5 cm diameter; 15 cm long), which forked intotwo arms (2.5 cm diameter, 7.5 cm long). Three B. cinerea-infected (5–10% or 30–60% or 90–100%) (6.89 ± 0.325 g;mean ± SE) and three (7.01 ± 0.152 g; mean ± SE) appropriatecontrol leaves of V. vinifera were enclosed in the glass cham-bers that were connected to the ends of the Y-tube by flexibleTeflon tubes. Compressed air with a flow rate of 300 mL/minwas allowed to enter the chamber through the Teflon tube inletand to come out through the outlet. Moths were introducedindividually through the downwind end of the Y-tube andobserved for 15 min. Moths that remained at the downwind endfor 30 min were deemed unresponsive and excluded from theanalyses. Female moths were also tested, but they did notrespond in the Y-tube, so were excluded from the analysis.

Larval bioassays

Two-choice experiment

This bioassay was conducted to test the preference of neonatelarvae of E. postvittana towards B. cinerea-infected (5–10% or30–60% or 90–100%) and appropriate control leaves by fol-lowing Foster and Howard (1999) with little modification. Inall experiments, newly hatched larvae were used (<2 h old). Inthis experiment, a glass tube (30 × 3 cm internal diameter)with an opening of 2 cm diameter in the centre was used. Onecontrol leaf and one B. cinerea-infected leaf were insertedindividually at each end of the tube, separated by 6 cm fromthe midpoint. Ten newly hatched larvae were gently placed inthe tube through the opening, which was immediately sealedwith Parafilm (Fig. 4). After 24 h, the position of the larvaewas noted. This experiment was repeated 10 times. The sameincubation conditions used in the two-choice experimentassaying the oviposition behaviour were used.

Larval acceptance and transmission of conidia of B. cinerea

This bioassay was conducted to test the acceptance and trans-mission of B. cinerea conidia following Foster and Howard(1999), and Bailey et al. (1997). An intensely infected (90–100%) B. cinerea-infected leaf with sporulating B. cinerea oran uninfected (control) leaf was placed in a 9-cm-wide Petriplate that included a damp filter paper. A neonate larva raisedfrom the egg stock from the refrigerated egg rafts was placed oneach leaf; the edges of each Petri plate set were sealed withParafilm (Fig. 5). After 72 h, either acceptance or mortality of

the larva was noted. To determine the presence of conidia of B.cinerea on the cuticle of larvae, each larva from control orB. cinerea-infected leaf was cleaned in 2-mL sterilised distilledwater in a vortex mixer (VM1, Ratek, Vic., Australia) (Baileyet al. 1997), and the washed water was examined in a lightmicroscope (YS2-H, Nikon, Tokyo, Japan). For internal exami-nation, each larva was surface sterilised with 10% NaClOsolution for 1 min and then washed with sterilised water (×3)and dissected in a stereo-binocular microscope (S-20, AISInstrument Services). Gut material of each larva was spreadover PDA plate and incubated at 22°C in dark for 72 h toobserve whether B. cinerea colonies were established. Theexperiment was repeated 30 times.

Statistical analysis

In the two-choice experiment, the numbers of eggs laid onB. cinerea-infected and appropriate control leaves were sub-jected to a paired sample t test. Number of eggs from theno-choice experiment and effect of volatile experiment wereanalysed using an independent sample t test. To get differentlevels of infection, differently aged leaves were required, so aseparate control was used for each infection level and thereforeindividual t tests were considered appropriate. Data from theY-tube olfactometer and choice of the female moths forB. cinerea-infected or control leaves were analysed using chi-square test. Analyses were conducted with SPSS statistic 17.0(1993–2007). Graphs were generated in MS Excel 2013.

RESULTS

Bioassay of oviposition behaviour

Two-choice experiment

In the two-choice experiment, when visual, olfactory andtactile sensory cues were available for E. postvittana adults,the females showed a significant preference for control leaves

Fig. 4. Custom-made glass device for the two-choice experi-ment of larvae of Epiphyas postvittana (not to scale). hgt,horizontal-glass tube; il, infected leaf; pe, port of entry; ps,parafilm seal; ul, uninfected leaf.

Fig. 5. Petri plate pair test for the larval acceptance and trans-mission of conidia of Botrytis cinerea (not to scale). dfp, dampfilter paper; pp, Petri plate; ul, uninfected leaf; il, infected leaf.

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(70%) compared with the intensely infected leaves (30%,n = 40, χ2 = 6.4, P = 0.01, Fig. 6). On the contrary, no signifi-cant difference in the preference for control leaves (45%)compared with mildly infected leaves (55%, n = 40, χ2 = 0.4,P = 0.53, Fig. 6) and for control leaves (63%) compared withthe moderately infected leaves (37%, n = 40, χ2 = 2.5,P = 0.11, Fig. 6) occurred.

For oviposition, mild infection of leaves did not significantlydeter the oviposition of E. postvittana (control vs. infected,437 ± 58 vs. 375 ± 63 (mean ± SE), t = 0.83, P = 0.47, Fig. 7),but the moderately and intensely infected leaves significantlydeterred oviposition (moderate infection, control vs. infected,451 ± 9.24 vs. 185 ± 62 (mean ± SE), t = 2.78, P = 0.03, Fig. 7;intense infection, control vs. infected, 599.3 ± 22 vs. 108 ± 37(mean ± SE), t = 9.89, P = 0.001, Fig. 7).

No-choice experiment

Gravid females oviposited on either B. cinerea-infected leaves(5–10% or 30–60% or 90–100%) or appropriate controlleaves. No egg occurred at the B. cinerea-infected sites onV. vinifera leaves. No significant difference occurred in thenumber of eggs laid on control leaves compared with themildly infected leaves (control vs. infected, 264.2 ± 20 vs.214 ± 16 (mean ± SE), t = 2.03, P = 0.08, Fig. 8). Signifi-cantly fewer eggs were laid on moderately and intenselyinfected leaves compared with the control leaves (moderatelyinfected, control vs. infected, 250 ± 35 vs. 85 ± 16 (mean ±SE), t = 4.19, P = 0.01, Fig. 8; intensely infected, controlvs. infected, 208 ± 27 vs. 36 ± 16 (mean ± SE), t = 5.841,P = 0.001, Fig. 8).

Effect of volatiles on oviposition

In a no-choice bioassay context, gravid females oviposited onthe smooth-surfaced, corrugated wall of the Dixie cup, in theresponse to the volatile compounds emitted from B. cinerea-infected leaves (5–10% or 30–60% or 90–100%) or appropri-ate control leaves. Volatiles from mild and moderate infectedleaves did not significantly deter the oviposition ofE. postvittana (mild infection, control vs. infected, 396 ± 10vs. 403 ± 16 (mean ± SE), t = −0.56, P = 0.58, Fig. 9; moder-ate infection, control vs. infected, 356 ± 16 vs. 339 ± 20(mean ± SE), t = 0.65, P = 0.53, Fig. 9). Significantly fewereggs were laid on the wall of cups that contained volatiles fromintensely infected leaves compared with the control leaves(control vs. infected, 357 ± 18 vs. 285 ± 26 (mean ± SE),t = 2.27, P = 0.04, Fig. 9).

Y-tube experiment

Male moths exhibited a significant level of preference for theodour of control leaves (67.5%) compared with intensely

Control

Control

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90–100%

30–60%

5–10%

80 8070 7060 6050 5040 4030 3020 2010 100

Percentage

*

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Fig. 6. Response of females Epiphyas postvittana in a ‘two-choice’ experiment towards uninfected (control) leaves ( ) andBotrytis cinerea-infected leaves ( ) of Vitis vinifera: 5–10% (a),30–60% (b), 90–100% (c). Levels of infection expressed in per-centage. *indicates a significant difference (*P < 0.05; NS, notsignificant).

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Fig. 7. Mean number of eggs laid by Epiphyas postvittana in a‘two-choice’ experiment with uninfected (control) leaves ( ) andBotrytis cinerea-infected leaves ( ) of Vitis vinifera: 5–10% (a),30–60% (b), 90–100% (c). Error bars denote standard error of themean (n = 8). Levels of infection expressed in percentage. *indi-cates a significant difference (*P < 0.05; ***P < 0.001; NS, notsignificant).

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Fig. 8. Mean number of eggs laid by Epiphyas postvittana in the‘no-choice’ experiment with uninfected (control) leaves ( ) orBotrytis cinerea-infected leaves ( ) of Vitis vinifera: 5–10% (a),30–60% (b), 90–100% (c). Error bars denote standard error of themean (n = 10). Levels of infection expressed in percentage. *indi-cates a significant difference (*P < 0.05; ***P < 0.001; NS, notsignificant).

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infected leaves (32.5%, n = 40, χ2 = 4.9, P = 0.03, Fig. 10). Onthe contrary, no significant difference occurred in the prefer-ence for control leaves (56.6%), compared with mildlyinfected leaves (43.4%, n = 40, χ2 = 0.53, P = 0.45, Fig. 10).No significant difference occurred in the preference for controlleaves (63.6%) compared with moderately infected leaves(36.6%, n = 40, χ2 = 2.13, P = 0.14, Fig. 10). Female moths(n = 10) did not respond to any treatment in this experiment.

Larval bioassays

Two-choice experiment

The neonate larvae of E. postvittana (n = 100) demonstrated asignificant preference for the mildly infected leaf (61%) com-pared with control leaf (39%, χ2 = 5.33, P = 0.02, Fig. 11) and

for moderately infected leaf (60%) compared with control leaf(40%, χ2 = 3.84, P = 0.049, Fig. 11). Whereas the larvaeshowed no significant difference in their preference whenallowed to choose either the intensely infected leaf (49%) orthe control leaf (51%, χ2 = 0.05, P = 0.82, Fig. 11).

Larval acceptance and transmission of conidia of B. cinerea

The introduced neonate larvae of E. postvittana on control leafor B. cinerea-infected leaf remained alive and active for thenext 72 h. External and internal body examination of the larvaefed on control leaf demonstrated that they did not carry anyconidia or hyphae of B. cinerea. Whereas, the larvae fed on theB. cinerea-infected leaf carried the conidia of B. cinerea ontheir body surface (Table 1). Internal examination demon-strated that the larvae fed hyphae and conidia of the fungus, asthese were found in the gut of the larvae. Conidia from the gutmaterial of all larvae, fed on infected leaves, tested positiveand resulted in the growth of B. cinerea on the PDA plate.

DISCUSSION

The test of preference patterns of E. postvittana indicates thatthe moderately and intensely B. cinerea-infected leaves ofV. vinifera significantly deter oviposition, whereas the larvae

0

50

100

150

200

250

300

350

400

450M

ean

num

ber

of e

ggs

*NS

NS

30–60% 90–100%5–10%(a) (b) (c)

Fig. 9. Mean number of eggs laid by Epiphyas postvittana oncorrugated wall of the Dixie cup, in response to the volatilecompounds emitted from uninfected (control) leaves ( ) orBotrytis cinerea-infected leaves ( ) of Vitis vinifera: 5–10% (a),30–60% (b), 90–100% (c) in ‘effect of volatile on oviposition’experiment. Error bars denote standard error of the mean (n = 10).Levels of infection expressed in percentage. *indicates a signifi-cant difference (*P < 0.05; NS, not significant).

Control

Control

Control

90–100%

30–60%

5–10%

80 8060 6040 4020 0 20

90-100

30-60

05-10%

Percentage

*

NS

NS

(b)

(a)

(c)

Fig. 10. Response of adult male Epiphyas postvittana in a‘Y-tube’ experiment towards uninfected (control) leaves ( ) andBotrytis cinerea-infected leaves ( ) of Vitis vinifera: 5–10% (a),30–60% (b), 90–100% (c). Levels of infection expressed in per-centage. *indicates a significant difference (*P < 0.05; NS, notsignificant).

Control

Control

Control

90–100%

30–60%

5–10%

80 8070 7060 6050 5040 4030 3020 2010 100

1

2

3

Percentage

NS

*

*

(a)

(b)

(c)

Fig. 11. Response of larvae of Epiphyas postvittana in a ‘two-choice’ experiment towards uninfected (control) leaves ( ) andBotrytis cinerea-infected leaves ( ) of Vitis vinifera: 5–10% (a),30–60% (b), 90–100% (c). Levels of infection expressed in per-centage. *indicates a significant difference (*P < 0.05; NS, notsignificant).

Table 1 Percentage acceptance of Botrytis cinerea-infected Vitisvinifera leaves by the larvae of Epiphyas postvittana and its per-centage that carried viable conidia of B. cinerea

Treatment Larvaeexamined

Acceptance(%)

Percentage of larvaecarrying conidia of

Botrytis cinerea

Body surface Gut

Infected leaf(90–100%)

30 100 100 100

Control leaf 30 100 0 0

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exhibit a significant level of preference for mildly (5–10%)and moderately (30–60%) B. cinerea-infected leaves but, onthe contrary, show no significant level of preference forthe intensely (90–100%) B. cinerea-infected leaves. Viableconidia occurred in the gut and on the cuticle of the larvae thathad fed on B. cinerea-infected leaves, thus indicating that thelarvae spread the conidia among the healthy leaves and berries.

Infection by fungi induces changes in the nutritional statusof the host plant, which in turn can also influence the volatilesemitted by the infected plant (Cardoza et al. 2002; Tasin et al.2012). These changes influence the olfactory response of theLepidoptera by either inhibiting or enhancing attraction and/oroviposition (Cardoza et al. 2003b; Dötterl et al. 2009; vanDam 2009; Tasin et al. 2011). In the present study, the decisionmade on the host quality for oviposition by the gravidE. postvittana was influenced by the stimuli that arose fromV. vinifera infected by B. cinerea. V. vinifera leaves infected byB. cinerea elicit avoidance behaviour in adult femaleE. postvittana and thus prevent oviposition. Although the mild(5–10%) level of infection by B. cinerea did not significantlydeter oviposition, the moderate (30–60%) and intense (90–100%) levels of infection significantly deterred oviposition.Avoidance of leaves of vascular plants infected by a fungalpathogen for oviposition by an herbivorous insect is an adap-tive strategy for the fitness of the offspring (Niinemets et al.2013). An infected plant could still be a source of food for theLepidoptera, as the associated fungus could improve insectnutrition either by breaking the complex materials into simplerforms or by decreasing the level of defence compounds(Hatcher 1995; Cardoza et al. 2003a). However, the time dif-ference between egg laying and hatching could increase thelevel of infection, and therefore, the potential larval food couldrapidly deteriorate in quality (Tasin et al. 2012). In such con-texts, the avoidance of moderately and intensely B. cinerea-infected sites on the leaves of V. vinifera by the ovipositingadults of E. postvittana suggests that it is an adaptive strategyof E. postvittana for ensuring suitable feeding sites for theiroffspring. Odours from leaves with an intense level of infec-tion (90–100%) also deterred the male moth, which couldpositively affect mating behaviour, as the male moths use plantvolatiles to distinguish environments where the likelihood tofind females is higher (Ansebo et al. 2004).

In the ‘no-choice’ bioassays, no eggs were laid at theB. cinerea-infected sites on V. vinifera leaves. E. postvittanalaid significantly fewer eggs on moderately (30–60%) andintensely (90–100%) B. cinerea-infected leaves, which rein-forces the findings made in the two-choice experimentreported in this paper. B. cinerea is known to degrade hydro-carbons and to release volatile compounds, such as 3-methyl-1-butanol, which is an indicator of decaying plant tissues(Magan & Evans 2000; Tasin et al. 2012). These physiologicalchanges consequent to tissue decay and the release of volatilesare sensed by the gravid E. postvittana, which is repelled, andin turn deterred from oviposition. Applying the preference-performance hypothesis (Gripenberg et al. 2010), we interprethere that the decayed quality of the leaves, which the larvaewould be encountering on hatching, would not be optimal for

their performance. A similar result was observed for gravidfemales of Lobesia botrana that oviposited on 1- to 3-day-old B. cinerea-infected berries of V. vinifera but significantlydeterred ovipositing on 7- to 9-day-old B. cinerea-infectedberries of V. vinifera (Tasin et al. 2012).

Tactile stimuli including surface texture play a secondaryrole in host selection, especially for oviposition (Foster &Howard 1998; Rojas et al. 2003). Depending on the character-istics of their host plants or the species-dependent optimalconditions for survival of the eggs, females prefer smooth,hairy or rough surfaces for oviposition (Renwick & Chew1994). Ovipositional behaviour in E. postvittana is stronglyinfluenced by tactile stimuli (Foster et al. 1997). The gravidE. postvittana prefers to oviposit on a smooth surface withvaricose texture rather than on rough and hairy surface.E. postvittana laid more eggs on the adaxial leaf surfaces thatbears fewer hairs (Fig. 12) than abaxial surface (Tomkins et al.1991). Where gravid E. postvittana were allowed to lay eggson smooth plastic Dixie cup with varicose structure in thepresence of fungi at mild, moderate and intense infectionlevels, only the intense level of infection (90–100%) deterredthe oviposition of E. postvittana significantly (P < 0.05). Thenumber of eggs laid on the Dixie cup at all infection levels(mild, moderate and intense) including the control leaves weregreater than the number of eggs laid on leaves at the sameinfection levels in a ‘no-choice’ bioassay (cf. Figs 8,9). Basedon this observation, we explain that the tactile stimuli mayinfluence oviposition in E. postvittana. The relative differ-ences between the number of eggs laid on control leaves andB. cinerea-infected leaves at all infection levels (mild (50.70),moderate (161.32) and intense (172.02) ) were higher in the‘no-choice’ bioassay (Fig. 8) compared with the ‘effect ofvolatile’ bioassay (mild (4.89), moderate (19.95) and intense(80.11) ) (Fig. 9). Therefore, we infer from these data that themycelial bed of B. cinerea, which is apparently similar totrichomatous leaf surfaces (Fig. 12), may influence theovipositional behaviour of E. postvittana. Our results supportthe findings of other studies, where it has been suggested thatthe texture and tactile features of the leaf surface are keytriggers factors and influence oviposition (Tomkins et al.1991; Foster & Howard 1998; Rojas et al. 2003). Chemicaland visual cues are likely to play an important role onovipositional behaviour of E. postvittana that needs to beexplored.

The larvae of E. postvittana showed significant attractiontowards mildly and moderately B. cinerea-infected leaves ofV. vinifera in the two-choice experiment, notwithstanding thefact the experiments have been done using excised leaves.However, no significant difference occurred when the infec-tion level was intense. This suggests that the larvae ofE. postvittana have mutualistic relationship with B. cinerea.Senescence of the infected leaves of V. vinifera releases nitrog-enous material for E. postvittana larvae, and this is accelerateddue to infection by B. cinerea. Similar results have beenreported for the larvae of L. botrana reared on the fruits ofV. vinifera and Pyrus malus (Rosaceae) infected by B. cinerea(Savopoulou-Soultani & Tzanakakis 1988; Mondy et al.

Effect of B. cinerea infection on E. postvittana 67

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1998a,b; Mondy & Corio-Costet 2000). The intensely infectedleaves of V. vinifera deteriorate more rapidly and that possiblyexplains why such leaves are unattractive to the larvae ofE. postvittana. Mutualistic relationship between insects andfungi on vascular plants is common among diverse insects(Svoboda et al. 1994; Mondy et al. 1998a), where the fungi actas a source of nitrogen, carbohydrates, vitamin B and sterols(Hatcher 1995).

Whether B. cinerea has a role in the development ofE. postvittana by acting as a food source or by supplementingnutrients and sterols remains to be verified. In the presentstudy, we used the neonate larvae of E. postvittana in experi-ments to test whether the B. cinerea-infected leaves were pre-ferred by them. All the larvae of E. postvittana were alive after72 h, feeding on B. cinerea-infected leaves indicating that thelarvae accepted B. cinerea as a part of their diet. Dissection ofthe first-instar larvae showed viable conidia in their gut, whichreinforces that the larvae do not exclude B. cinerea duringfeeding on the leaf tissues of V. vinifera. Moreover, the larvaetransport viable conidia on their body surface, thus distributingthe fungus to new sites on the plant. The results of the presentstudy are similar to those of Bailey et al. (1997), who foundviable conidia of B. cinerea on the cuticle, in the gut and thefaeces of the fourth-instar larvae of E. postvittana.

In this study, we show that the gravid females ofE. postvittana avoid B. cinerea-infected leaves duringoviposition and the rate of oviposition are inversely related tothe levels of infection. On the contrary, the larvae ofE. postvittana prefer to feed on mild and moderatelyB. cinerea-infected leaves. Incidence of viable conidia ofB. cinerea in the gut and on the body surface of the larvae of

E. postvittana suggest that E. postvittana plays a role inspreading the conidia of B. cinerea on the leaves of V. vinifera.The co-occurrence of the B. cinerea and larvae ofE. postvittana on V. vinifera and the preference of the larvae ofE. postvittana towards B. cinerea-infected leaves indicate apossible mutualism, which remains to be verified.

ACKNOWLEDGEMENTS

Aaron Simmons (Department of Primary Industries, Orange,NSW, Australia) read the prefinal draft of this paper andoffered useful and insightful comments. We are grateful toMichael Priest (Department of Primary Industries, Orange,NSW, Australia) for identifying fungal materials and to FengYi (Waite Campus, The University of Adelaide, Adelaide, SA,Australia) for supplying eggs of E. postvittana.

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Accepted for publication 1 March 2014.

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