Arthropod-Plant Interactions

, Volume 5, Issue 2, pp 149–161

The response of resistant kiwifruit (Actinidia chinensis) to armoured scale insect (Diaspididae) feeding

Authors

    • The New Zealand Institute for Plant and Food Research Limited
  • N. A. Mauchline
    • The New Zealand Institute for Plant and Food Research Limited
  • M. K. Jones
    • The New Zealand Institute for Plant and Food Research Limited
  • P. W. Sutherland
    • The New Zealand Institute for Plant and Food Research Limited
Original Paper

DOI: 10.1007/s11829-011-9124-9

Cite this article as:
Hill, M.G., Mauchline, N.A., Jones, M.K. et al. Arthropod-Plant Interactions (2011) 5: 149. doi:10.1007/s11829-011-9124-9

Abstract

The responses of five experimental genotypes and one commercial variety of kiwifruit (Actinidia chinensis) to attack by two polyphagous, congeneric armoured scale insect pests (Hemiberlesia rapax and H. lataniae) are described. H. lataniae feeding elicits a response in the bark and fruit of all but one of the experimental genotypes, leading to the development of wound periderm over a 4–5 week period, and death of the insect. The response, which differs slightly between tissue types and genotypes, consists of wound periderm formation in a bowl shape beneath and around the insect, preventing its stylet from reaching normal unmodified parenchyma tissue. Wound periderm cell walls become suberised and cells beneath the insect become filled with phenolic compounds. In some cases, cells beneath the insect become hypertrophic or undergo lysis, exhibiting characteristics of a hypersensitive-like response. The remaining genotype showed no physical change in tissue structure in response to H. lataniae feeding, and the insects survived but were substantially reduced in size. These results suggest that both physical and chemical plant resistance responses are involved. In contrast, H. rapax elicited no observable histological response from any of the genotypes and the insects developed normally on bark and fruit. Both insect species developed normally on leaf petioles and these exhibit only slight cell wall thickening in response to their feeding. This unusual plant defensive response to a sucking insect has similarities to simple types of gall formation in response to insect and pathogen attack and has characteristics of resistance gene-mediated models of plant defence.

Keywords

Plant resistanceWound peridermHypersensitive responseDiaspididaeArmoured scale insectsGalls

Introduction

In spite of their economic importance as pests of perennial horticultural crops, the interactions between armoured scale insects (Homoptera: Diaspididae) and their hosts have received very little study (Miller and Davidson 2005). By contrast, at the next level of trophic interaction, armoured scale insect host-parasitoid interactions and biological control have been the subjects of a large body of basic and applied research (Rosen 1990; Miller and Davidson 2005). The spectacular success of classical and augmentative biological control in regulating populations of several key diaspidid pest species on horticultural crops in the middle and late decades of the twentieth century has led to many being successfully controlled without recourse to studies of host plant resistance (Rosen 1990).

This study of the interaction between armoured scale insects and their hosts arises from the need for economic control of two armoured scale insect pests, greedy scale (Hemiberlesia rapax Comstock) and latania scale (Hemiberlesia lataniae Signoret) on kiwifruit in New Zealand, and the absence of effective biological control options. These insect species are both highly polyphagous and occur throughout the world in tropical to temperate zones (Miller and Davidson 2005; Normark and Johnson 2010; CABI 2009). Both species exist in New Zealand as uniparental, parthenogenetic populations, having two generations per year and are resident on the wood of kiwifruit vines throughout the year, settling on leaves and fruit during the summer (Edwards et al. 2008).

A research programme to investigate kiwifruit resistance to armoured scale insects has found large differences in the levels of armoured scale insect infestation on canes and fruit of germplasm from the species Actinidia chinensis Planch. with some genotypes showing complete resistance to H. lataniae, but not to H. rapax (Hill et al. 2009). During the course of these studies, swelling was noticed around the insects on the canes of some resistant genotypes, followed by death of the insects. This study investigates this unusual response of several A. chinensis genotypes to feeding by H. lataniae and H. rapax. The response of the cane bark, fruit and petiole to feeding by H. lataniae and H. rapax is described. We suggest that this is an example of a gene-for-gene mediated plant resistance response. We hypothesise, with reference to the insect gall-formation literature, that the histological response of the A. chinensis varieties to H. lataniae is characteristic of a simple insect gall and may represent a precursor to the evolution of true galls, a hypothesis proposed 80 years ago by Zweigelt (1931). An understanding of the interaction between these pest species and kiwifruit germplasm will assist the breeding of future commercial varieties with enhanced levels of resistance.

Methods

Plant material

The plant material consisted of canes, leaves and fruit on 10-year-old vines of the commercial kiwifruit variety A. chinensis ‘Hort16A’ and 12-year-old vines from four experimental A. chinensis genotypes (2C, 3C, 8A, 9A) from a breeding experiment (details in Cheng et al. (2004)). All vines were diploid. Previous research (Hill et al. 2007) had shown that these genotypes exhibited resistance to H. lataniae but not to H. rapax. All experiments were carried out on vines growing in orchard blocks in the field, at the Plant & Food Research Te Puke Research Centre, New Zealand.

Insect settlement assay

Twenty Hemiberlesia lataniae and H. rapax crawlers (the newly eclosed, mobile stage of the first instar) were settled separately using a fine brush, onto five leaves of four vines of each of the five genotypes on 23–24 November 2006. Twenty crawlers of each species were also settled in the same manner onto sites on new season (<1 year old (yo)) and previous season (>1 yo) canes on five vines of each genotype on 10 and 11 January (H. lataniae) and 15 and 17 January 2007 (H. rapax). Wool was wrapped around the cane to aid crawler settlement. The wool was removed on 18 January 2007. Crawlers of both scale insect species were settled onto fruit in the same manner on 8–10 March 2007. Observations of scale insect settlement and plant response began as soon as the insects were settled on the leaves or fruit and as soon as the wool was removed from the bark. The insects were photographed using a Canon Coolpix 995 camera with a frame attached to the front allowing the size of the insect to be measured in the image (Hill et al. 2005). Observations of the scale insect nymphs and the plant tissue around them were made three times a week for 5–7 weeks until no further changes were observed. Any physical response by the plant in the form of blackening or swelling of tissue around or beneath the scale was recorded.

Tissue samples for histology, including the insect, were taken on days 14, 21, 28 and 35 after crawler settlement for cane samples, day 41 for leaf (petiole) and days 42 and 49 for fruit samples in 2007/2008. A second series of cane (<1 yo) and fruit samples with settled H. lataniae nymphs was collected at weekly intervals for 5 weeks after crawler settlement from genotypes ‘Hort16A’ and 2C in November/December 2007 (cane samples) and March/April 2008 (fruit) for microscopic examination. Finally bark samples were taken 5 weeks after H. lataniae crawler settlement on >1 yo canes of a partially resistant experimental A. chinensis variety ‘Hort22D’ in January 2010 to allow comparison of the histological response between completely and partially resistant germplasm.

Microscopy

Pieces of tissue (fruit, canes and leaves) 5mm × 5 mm square were fixed in 2% (v/v) formaldehyde with 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer pH 7.2 under vacuum for 1 h, washed in buffer, dehydrated with an alcohol series, and embedded in LR White resin (London Resin Company Ltd, Reading, England) (Sutherland et al. 2004). Embedded material was sectioned with a diamond knife on a Leica UCT ultramicrotome at a thickness of 1 μm and stained with 0.05% (w/v) toluidine blue in borate buffer (pH 4.4). Light microscopy was carried out on an Olympus Vanox AHT3 compound microscope (Olympus, Tokyo) with image recording by a CoolSnap digital camera (Roper Scientific, Tucson, AZ). Forty-two insects and their attached substrate were sectioned, stained and mounted (details in Online resource 1) and over 500 slides were prepared and examined.

To visualise autofluorescence of suberin/lignin and condensed phenolics, the UV filter (excitation 330–385 nm, emission 420 nm) and the G (green) filter (excitation 545 nm, emission 590 nm) were used respectively on 1-μm thick sections (Ruzin 1999). In our experience glutaraldehyde-induced autofluorescence is minimal at this thickness. For immunolabelling with JIM5 (anti-homogalacturonan; unesterified or partially esterified) (Clausen et al. 2003), 200-nm thick sections were mounted on poly-l-lysine coated slides and dried overnight. They were then blocked with 0.1% bovine serum albumin (BSA-c, Aurion, Wageningen, The Netherlands) in PBS-T (phosphate-buffered saline plus 0.1% Tween® 20) for 15 min and then incubated overnight in JIM5 diluted 1:20 in blocking buffer. After incubation the slides were washed in PBS-T and incubated in goat anti-rat Alexa 488 diluted 1:600 in PBS. Sections were finally washed in 2–3 ml PBS-T and mounted in Citifluor AF1 antifadent solution (Citifluor, Leicester, UK). For details of staining techniques for Actinidia tissue and a description of Actinidia fruit skin histology, see Hallett and Sutherland (2005).

Measuring scale insect size

The size of the scale insect cap on a minimum of 10 scale insects per substrate was estimated using the image analysis software ImageJ (version 1.37v; http://rbs.info.nih.gov/ij/) from the photographic images taken in the field (Hill et al. 2005) at a range of dates up to 6 weeks after crawler settlement.

Results

Armoured scale insect settlement, survival and growth

Initial numbers of scale insect crawlers settling on fruit was the same for the four experimental genotypes (Mann–Whitney test on each genotype and on data from all genotypes combined; P > 0.1), but H. lataniae settlement on ‘Hort16A’ fruit was much lower than that of H. rapax (Mann–Whitney test P < 0.001; Table 1). At the end of the experiment, 43 days after crawler settlement onto fruit, only 3.6% of the originally settled H. lataniae nymphs were still alive compared with 54% of settled H. rapax (Table 1). Some of the dead scale insects remained stuck to the fruit, but most of them had fallen from the fruit surface. At the end of the experiment, of the scale still on the fruit, most H. rapax were alive and in the third instar, while most H. lataniae were dead first or second instars and the remaining live H. lataniae were all in the second instar (Table 2).
Table 1

Total number of armoured scale insects on fruit 8–11 days after crawler settlement and the number of scale insects and proportion that were alive on the fruit 43 days after crawler settlement

Genotype

No. scale settling after 8–11 days

Survival to day 43

Total scale on fruit

% Live scale

H. lataniae

H. rapax

H. lataniae

H. rapax

H. lataniae (%)

H. rapax (%)

‘Hort16A’

8

78

4

78

37.5

87.2

2C

105

82

26

49

1.9

51.2

3C

53

66

5

42

1.9

39.4

8A

70

72

11

41

4.3

50.0

9A

72

85

8

42

2.8

42.4

Total

308

383

54

252

3.6

54.3

Table 2

The distribution of life stages of live and dead H. lataniae and H. rapax after 43 days on fruit from all vines

 

Live

Dead

Total

1st instar

2nd instar

Adult

1st instar

2nd instar

Adult

H. rapax

No.

0

42

164

3

21

22

252

%

0%

17%

65%

1%

8%

9%

 

H. lataniae

No.

0

11

0

17

26

0

54

%

0%

20%

0%

31%

48%

0%

 
Preliminary tests (one-way ANOVA comparing genotypes within scale ages) showed there were no differences (P > 0.05) in the sizes of the armoured scale insect caps between genotypes and data from all vines were pooled within tissue-type to compare the effect of tissue on armoured scale insect species growth. On canes and fruit, the scale caps of H. rapax nymphs grew in size exponentially (canes: y = 0.1171 + 0.000001064exp(0.3676x); r2 = 0.97), whereas H. latania caps did not grow at all on canes or fruit (Fig. 1). By contrast, there was no difference in the growth of H. rapax and H. lataniae on petioles (Fig. 1).
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Fig. 1

Growth of Hemiberlesia rapax (solid circle and line) and H. lataniae (open circle and dashed line) for 6 weeks after crawler settlement on canes, fruit and petioles of A. chinensis ‘Hort16A’. Vertical bars are 95% confidence intervals

Externally visible response of the plant to scale insect settlement and feeding

Canes

Hemiberlesia rapax settled on the canes elicited no externally visible response from any of the vines. Within 2 weeks of H. lataniae settlement on the new canes (<1 yo), the bark tissue around the canes had begun to show a response in the form of a swelling of the tissue (neoplasm) immediately around the insect. The progression of the response within the scale insect population increased steadily over a 6-week period until over 90% of the scale insects on the new canes of four of the five genotypes had elicited a recognisable response by the plant when viewed from above (Fig. 2). The plant response was generally slower to appear and less pronounced on the old (>1 yo) than on the new canes (Fig. 2), although there was no difference in the response with cane age in ‘Hort16A’. Genotype 8A responded slightly differently from the rest, with fewer than 50% of the insects eliciting a visible plant response.
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Fig. 2

Percentage of H. lataniae insects eliciting a visible surface response on canes over time for four experimental A. chinensis genotypes and the variety ‘Hort16A’. A 3-parameter sigmoid equation (y = a/(1 − exp(−(x − x0)/b))) is fitted to the data for each plot. Circles and solid lines = young (<1 year old (yo)) canes, triangles and dashed lines = old (>1 yo) canes

On the <1 yo canes, the tissue immediately surrounding the insect blackened, indicating localised cell death (hypersensitive-like reaction). After 6 weeks, the swelling and cell death of plant tissue adjacent to the insects was very pronounced, especially in genotypes 2C, 3C and ‘Hort16A’ (Fig. 3).
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Fig. 3

Neoplasm (tissue swelling) and cell death on new (<1 year old (yo)) A. chinensis canes around H. lataniae nymphs 5 weeks after crawler settlement. a variety ‘Hort16A’, b genotype 2C, c genotype 3C. The white scale insect caps have an approximate area of 0.07–0.1 mm2 (diameter ~0.3 mm)

Fruit

Hemiberlesia rapax on the fruit elicited no externally visible response from any of the vines. H. lataniae elicited no externally visible response from the fruit of any of the genotypes except for one vine of genotype2C which showed cell death (hypersensitive-like reaction) of the tissue around the scale 6 weeks after scale insect settlement.

Leaves

Crawlers of both species settled predominantly (95%) on the upper surface of the petiole at its junction with the leaf lamina (the remainder settling on the adaxial leaf lamina). After 6 weeks, the aggregations of both H. rapax and H. lataniae elicited a slight darkening of the petiole cells on genotype 2C only. No other visible leaf response to either species was observed.

Stylet penetration and feeding

During the course of cane bark sectioning to examine the plant response to the insect, sections including the stylet were encountered on several occasions. Stylet penetration was intra-cellular, moving more or less straight down through the periderm and collenchyma into the parenchyma where most of the feeding appeared to be taking place (Fig. 4; see Fig. 5 and Online resource 2 for labelling of tissue types). The stylets were not observed penetrating through the fibre layer (light blue thick-walled (lignified) cells beneath the parenchyma, or into the phloem which lies below the fibre layer in Fig. 4a.
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Fig. 4

Stylet paths within A. chinensis ‘Hort16A’ canes (<1 yo). a Five week old H. rapax stylet (arrowed) travelling intra-cellularly through the periderm (light blue cells), collenchyma (densely-packed more regular shaped and thicker-walled cells) and parenchyma (loosely packed and irregularly shaped cells) where it appears to be feeding on cell contents. b Higher magnification of 5 weeks old H. rapax stylet. c Five weeks old H. rapax stylet track ending at the base of the parenchyma next to the fibre layer. d One week old H. lataniae stylet (note: the insect’s cap has been displaced during preparation). Scale bars: a and b = 100 μm; c = 10 μm; d = 50 μm. (Color figure online)

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Fig. 5

Response of A. chinensis ‘Hort16A’ cane bark (<1 yo) to H. lataniae settlement and feeding 2–5 weeks after scale insect settlement. a 2 weeks: Np = Native periderm, C = collenchymas, Pa = parenchyma, F = fibre layer. Inset shows cells that will form the wound periderm beginning to divide. The insect has been dislodged during preparation, the arrow points to its original location. b 3 weeks: Wound periderm now well developed. c 4 weeks: Np = native periderm, Wp = Wound periderm. d 5 weeks: the double-headed arrow shows the approximate delimitation of the now fully–developed wound periderm (Wp). Many of the cells external to the wound periderm are full of dark-staining phenolic material and collapsing/dying cells. Scale bars = 100 μm

Histology of the plant response to armoured scale insect feeding

Cane bark

One week after H. lataniae settlement on ‘Hort16A’ cane bark, there was no evidence of a change in the plant cell structure (Fig. 4d). Two weeks after scale insect settlement (Fig. 5a), cell division had been initiated in collenchyma and parenchyma cells beneath and around the insect extending up to the periderm. The periderm layer beneath the insect (dislodged during preparation—its position is indicated by an arrow) also appeared to be undergoing an enlargement in cell size, becoming more like collenchyma cells in appearance. Three weeks (Fig. 5b) after scale insect settlement, an organised layer of cells characteristic of wound periderm (Evert 2006) had formed in a bowl-shape, encircling the insect. The wound periderm layer, about 8–12 cells thick, extended from the original (‘native’) periderm, beneath and at some distance from the insect, down through the parenchyma as far as the layer of fibre cells. Four weeks after scale insect settlement (Fig. 5c), the new cells making up the wound periderm (Wp) formed a continuous and morphologically indistinguishable layer with the original native periderm (Np) layer.The periderm layer beneath the insect had become indistinguishable from collenchyma, although the epidermis remained unmodified. Some of the cells in the collenchyma layer above the wound periderm were full of dark-staining phenolic material, while other cells had become hypertrophic or were showing signs of lysis and collapse. After 5 weeks (Fig. 5d), the bowl of wound periderm had undergone further cell division, forming a layer of cells up to 20 cells thick, completely encircling the scale insect, which was dead. Dark-staining phenolic material filled many of the cells between the wound periderm and the insect, and most of the remaining cells showed signs of lysis or collapse.

The response of genotypes 3C and 9A to H. lataniae after 5 weeks is similar to ‘Hort16A’. Viewed under autofluorescence (Figs. 6 and 7) and immunolabelling (Fig. 7) the sections show extensive deposition of suberin/lignin in the cell walls and middle lamellae (blue) of the collenchyma cells within the wound periderm ‘bowl’ (Fig. 6e). Suberin deposition in native and wound periderm is visualised in Fig. 7b by a dark layer, resulting from lack of antibody binding. Suberin deposition in the wound periderm appears to be restricted to the wound phellem (Figs. 6e and 7a; see also online resource 4), which is the case in potato tubers (Sabba and Lulai 2002). Phenolic material stains red in collenchyma cells between the wound periderm and insect (Fig. 6c), with some again showing lysis and cell collapse, while further phenolic deposition is evident in the wound periderm (Fig. 6c).
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Fig. 6

Transverse section of <1 yo cane bark from A. chinensis genotype 3C, a Five weeks after H. lataniae crawler settlement. c showing the distribution of phenolic fluorescence (red). e showing suberin/lignin (blue) fluorescence. Figures b, d and f show corresponding unchallenged bark. Autofluorescence overlay of images captured with G and UV filters (excitations and emissions in methods section). Scale bars = 100 μm. (Color figure online)

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Fig. 7

Response of A. chinensis genotype 9A <1 yo cane bark 4 weeks after H. lataniae settlement. a Autofluorescence overlay of images captured with G and UV filters (excitations and emissions in methods section): phenolics (red), suberin/lignin (blue). b Immunolabelling: pectin (green). Zone of reduced pectin labelling in native and wound periderm (due to suberin deposition) has been arrowed. Scale bars = 100 μm. (Color figure online)

The cane response to H. rapax offered a complete contrast to that observed for H. lataniae. There was no visible tissue response from any of the genotypes (Fig. 8), and the insect developed normally. After 4 weeks and particularly after 6 weeks, there was evidence of damage or disorganisation amongst the parenchyma and collenchyma cells beneath the insect, but it was unclear whether this was a plant response to feeding or damage caused by the insect feeding (Fig. 8b, c). There appeared to be many empty cells in the parenchyma tissue after 6 weeks, by which time the insect had moulted twice to become an adult. The section of cane of the partially resistant variety A. chinensis ‘Hort22D’ 5 weeks after H. latania settlement showed no signs of change to cell wall structure (Fig. 9). The insect was alive but only one-fifth of the size of a scale growing on a susceptible cane (Hill et al. 2011).
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Fig. 8

A. chinensis genotype 9A <1 yo bark a without insect feeding, b after 4 weeks of H. rapax feeding (note stylet track (arrowed)) and c after 6 weeks of H. rapax feeding. Scale bars = 100 μm

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Fig. 9

Longitudinal section of H.lataniae 5 weeks after crawler settlement on bark of >1 yo cane of partially resistant variety A. chinensis ‘Hort22D’, showing lack of a physical response by the plant tissues. Scale bar = 100 μm

Fruit

The fruit tissue did not respond to H. rapax feeding (Fig. 10b, d, f). The response of A. chinensis ‘Hort16A’ fruit to H. lataniae feeding after 6 weeks was similar to that observed for ‘Hort16A’ canes, with the formation of a ‘bowl’ of wound periderm 3–8 cells thick beneath the insect (Fig. 10a). The insect was dead. The response was less pronounced than for the canes, with little or no external swelling and little evidence of hypertrophy of the cells within the wound periderm bowl. The autofluorescence images for H. lataniae on A. chinensis ‘Hort16A’ (Fig. 10c–f) show, as with the cane images, suberin/lignin thickening of cell walls of the wound periderm cells and extensive deposition of phenolic compounds within the wound periderm cells and cortical cells beneath and around the insect. There was no obvious cell death. The response of genotype 2C fruit to H. lataniae feeding was quite different from that of ‘Hort16A’ (Fig. 11). Wound periderm formation was again evident, but in a reduced form and very close to the insect compared with the response in ‘Hort16A’. Cells immediately below and around the insect showed signs of phenolic deposition and cell death (Fig. 11). This appeared to resemble a hypersensitive-like reaction, characterised by death and collapse of the cells beneath and immediately around the insect (Fig. 11c), which was visible externally. The end result of the plant responses to H. lataniae in all the observed cases was insect death, after little or no discernible growth. Genotype 2C fruit showed some variability in the extent of its responses (see online resource material 5 and 6).
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Fig. 10

Response of A. chinensis ‘Hort16A’ fruit to H. lataniae (a, c, and e) and H. rapax (b, d, and f) 6 weeks after scale insect settlement. a and b toluidine blue staining. c and d condensed phenolic autofluorescence (red). e and f suberin/lignin autofluorescence (blue). The large thick-walled lignified cells are brachysclereides (stone cells). Scale bars = 100 μm. (Color figure online)

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Fig. 11

Response of A. chinensis genotype 2C fruit to H. lataniae 4 weeks after crawler settlement. a toluidine blue staining, b condensed phenolic autofluorescence (red). c suberin/lignin autofluorescence (blue). The large, thick-walled heavily stained cells in a and c are brachysclereids (stone cells) Scale bars = 100 μm. (Color figure online)

Petiole

Sections through the petiole (Fig. 12) showed slight thickening of the cell walls in the collenchyma/parenchyma in both H. rapax and H. lataniae-challenged tissue. Scale insects of both species continued to grow on the petioles up to the end of the experiment, 7 weeks after scale settlement (Fig. 1).
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Fig. 12

The response after 6 weeks, of petiole tissue from A. chinensis genotype 2C. a without insect challenge. bH.rapax feeding. cH. lataniae feeding. Note the slight thickening of the collenchyma cell walls (arrowed) visible externally as a darkening of the tissue beneath the scale insects, in the H. rapax and H. lataniae challenged petioles. Scale bars = 100 μm

Discussion

As far as we are aware, this is the first detailed description of a plant defensive response to a diaspidid scale, and the first record of wound periderm formation and hypersensitive–like reaction as a defensive response to diaspidid feeding. The response of cane and fruit tissue of the ‘resistant’ A. chinensis selections to H. lataniae settlement and feeding shows a consistent pattern of wound periderm formation in a bowl of suberised/lignified cells filled with phenolic material, effectively walling-off the insect from unmodified parenchyma tissue on which it can feed. There was some variability in the degree of cell hypertrophy and lysis (hypersensitive-like cell death) within the ‘bowl’ which may have been related to tissue age or genotype. The fruit tissue response was more variable than the cane bark tissue, and this may reflect physiological changes taking place in the rapidly-growing fruit. We did not set out to characterise fully the effect of tissue age on its response to insect feeding and the limited number of insects sectioned in this study means that we may not have observed the full range of plant responses to H. lataniae. The mode of insect death cannot be determined with certainty from the observations, however there is evidence to suggest that both physical (lignin/suberin deposition, cell lysis and collapse (e.g. Figs. 3, 6e, and 7a)) and chemical (phenolic deposition (e.g. Figs. 5d, 6c and 11b)) defensive mechanisms are involved. The observation of substantially reduced insect growth on the partially resistant genotype ‘Hort22D’ without a visible physical response from the plant (Fig. 9) would also suggest in this instance a predominantly chemical basis to the plant response.

The early initiation (within 2 weeks of H. lataniae settlement) of wound periderm formation through parenchyma cell division (Fig. 5a) in the absence of any other visible plant response or tissue change suggests that this is a primary response from the plant to the detection of the insect’s stylet. The later development (within 4–6 weeks) of a hypersensitive-like response in tissues between the wound periderm and the insect might indicate that this is a secondary response from the plant, possibly resulting from the wound periderm formation. However, there is ample evidence from studies of hypersensitive-like responses to fungal and bacterial invasion in plants to suggest that hypersensitivity is a major defensive response in its own right, mediated in many cases by specific resistance genes (Stuible and Kombrink 2004; Greenberg and Yao 2004). Thus, these two responses—hypersensitivity and wound periderm production—are most likely to be direct and distinct plant responses to insect attack. We hypothesise that both may be mediated by resistance genes in the manner of the gene-for-gene model of plant defence (Kaloshian 2004).

Wound periderm formation, cell hypertrophy and lysis (hypersensitive-like response), cell-wall lignin/suberin deposition and within-cell phenolic deposition, have been observed in previous studies of conifers, fruit and nut trees in response to invasion by fungi (Biggs 1989; Solla et al. 2002; Hoff and McDonald 1972), nematodes (Yamada and Ito 1993; Kaplan and O’Bannon 1981), a bark beetle (Franceschi et al. 2000) and a margarodid (Liphschitz and Mendel 1989). In most cases, these studies have noted a differential response between ‘resistant’ and ‘susceptible’ varieties, and the speed of wound periderm formation appears to be related to the degree of resistance in pathogen attacks. Most wound periderm responses occurred over a 3–5 week time frame, similar to those observed here. Also similar to the finding in this study that petiole, fruit and cane bark tissues mount different responses to H.lataniae feeding, Liphschitz and Mendel (1989) noted that wound periderm and hypersensitive-like responses occurred in the stem but not in the shoot cortex of resistant Pinus spp. challenged by Matsucoccus josephi Bodenheimer and Harpaz. The well known resistance of north American native Vitis rootstock to phylloxera (Daktulosphaira vitifoliae Fitch) in grapes is, as far as we are aware, the only other well documented example of hypersensitive-like response to feeding in the Coccoidea (Roush et al. 2007; Blank et al. 2009; Dietrich et al. 2009). The resistance of specific parts of the plant to H. lataniae fruit and bark, but not leaves, may not be unusual, as many diaspidids are known to feed on only one plant part (Beardsley and Gonzales 1975; Miller and Davidson 2005).

While the speed of wound periderm formation may be important in determining the degree of resistance in pathogen-tree interactions (Solla et al. 2002), for a slow-developing sessile insect like H. lataniae (2 generations per year, 8–12 weeks from eclosion to the onset of reproduction on kiwifruit in New Zealand (Hill et al. 2009)), wound periderm formation and a hypersensitive reaction would appear to be an ideal plant defensive response from which, unlike a pathogen, the insect cannot escape. Although this may be the first time that such a response has been noted against a diaspidid, there are over 30 published papers on host plant resistance in at least 16 economic plant species to 19 diaspidid species (MG Hill unpublished). In only one other instance, the tea scale (Pseudaulacaspis pentagona) (Mizuta 2003, 2005; Tanaka and Taniguchi 2007), has the underlying defence mechanism been studied in detail and a resistance gene located. Relative to other economically important homopteran families (e.g. Aphididae, Aleurodidae, Cicadellidae), host plant resistance to the Diaspididae (indeed to the Coccoidea) has received scant attention. The wound periderm defence responses and hypersensitive-type reactions in A. chinensis discovered in this study would suggest that closer examination of insect neonate -host interactions may provide a fruitful avenue for investigating plant defensive responses to Coccoidea. We suggest that the plant response observed here may be relatively common as a defence against the Diaspididae, and possibly more broadly, the Coccoidea, but because it acts upon first instars and can confer complete resistance to the insect species, may have been overlooked.

Actinidia response to scale insect feeding and its similarity to gall formation

Thirty-nine diaspidid species out of 2,370 (1.6%) have been recorded as gall-forming, with at least 12 showing stem or twig swellings (Gullan et al. 2005). The superficial similarity of the plant defensive responses observed in this study to those involved in gall formation by diaspidid (Larew 1990), asterolecaniid (Gullan et al. 2005) and cynipid (Harper et al. 2004) gall formers and to a gall-forming maize smut (Callow and Ling 1973), namely hypertrophy, hyperplasia, dense-staining cytoplasm, cell collapse and lignification, supports an old hypothesis that gall formation is a plant defensive reaction against herbivory that has come under the control and manipulation of the insect (Hilker and Meiners 2006; Zweigelt 1931).

A hypersensitive reaction to gall-forming insects is widespread across many plant families (Fernandes 1990; Fernandes and Negreiros 2001; Cornelissen et al. 2005). Gall formation can be host-dependent, tissue-dependent and within some genera, scale insect species-dependent (Larew 1990; Gullan et al. 2005), indicative of an insect-host interaction that is similar in principle to a gene-for-gene mediated plant defensive response.

Diaspidid feeding

Stylet penetration was intra-cellular and feeding appeared to occur primarily from the parenchyma. Diaspidid feeding has not received much study, but intra-cellular stylet penetration terminating in parenchyma has been observed previously in Pseudaulacaspis pentagona (Targ. Tozz.) on mulberry bark (Yasuda 1979) and by Aonidiella aurantii (Maskell) on citrus leaves and bark (Washington and Walker 1990). Stylet penetration into vascular tissues has also been observed in at least two instances, although it is probably not a main source of food (Heriot 1934; Washington and Walker 1990). This phenomenon was not observed in the current study, where the fibre layer of sclerotised cells beneath the parenchyma is likely to act as a barrier to stylet penetration into the vascular bundle.

The levels of host plant resistance observed in this study indicate that the development of future commercial kiwifruit varieties with enhanced levels of diaspidid scale insect resistance is a realistic prospect. Further work will seek to identify the genetic basis of kiwifruit resistance to armoured scale insects, and to understand the nature of the wound periderm response to the highly polyphagous H. lataniae but not to the congeneric and equally polyphagous H. rapax.

Acknowledgments

We are indebted to Margaret McCully, CSIRO, for advice on interpretation of the histological responses observed. Erik Rikkerink, Graham Walker and an anonymous reviewer provided comments on earlier drafts. The New Zealand Foundation for Research Science and Technology (C06X0301) provided funding support.

Supplementary material

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© Springer Science+Business Media B.V. 2011