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Home , Agromyzidae, Liriomyza trifolii, Serpentine leaf miner

Diagnostic Protocols for the Detection of Leafminers *****

Diagnostic Protocol for the Detection of the American Serpentine Leafminer, trifolii (Diptera: )

2005

Prepared by Mali Malipatil and John Wainer Department of Primary Industries Victoria, PMB 15, Ferntree Gully DC, Victoria 3156

The current version of this document is in draft form. All attempts have been made to ensure the scientific and technical content is correct, however independent verification of the information should be sought prior to use.

The material contained in this publication is produced for general information only. It is not intended as professional advice on any particular matter. No person should act or fail to act on the basis of any material contained in this publication without first obtaining specific, independent professional advice. Plant Health Australia and all persons acting for Plant Health Australia in preparing this publication, expressly disclaim all and any liability to any persons in respect of anything done by any such person in reliance, whether in whole or in part, on this publication. The views expressed in this publication are not necessarily those of Plant Health Australia.

1. Diagnostic images of American serpentine leafminer

See Figures 1 & 2.

2. Diagnostic images of Australian species with which American serpentine leafminer might be confused

See Figures 3 – 5.

3. Organisms in Australia with which American serpentine leafminer might be confused

Family Agromyzidae - The family Agromyzidae, of which the world fauna has about 1900 species in 26 genera, is represented in Australia by 13 genera and about 150 species. Of the 376 species in the genus Liriomyza, only 18 species occur in Australia (Spencer, 1977).

The following combination of characters will define the family Agromyzidae - Vibrissae present (Figure 6), 1-7 frontal bristles present (Figure 6); wing with costal break present at the apex of Sc, cell cup small, A1 not reaching wing margin (Figure 9); male with pregenital sclerites with a fused tergal complex of tergites 6-8, with only two spiracles between tergite 5 and genital segment; female anterior part of abdominal segment 7 forming an oviscape.

In practice, agromyzids are reasonably easy to recognize because the larvae of all species feed in the living plant tissue and approximately three-quarters of the species are leaf-miners. Generally the larvae are cylindrical in shape, tapering anteriorly, with projections bearing the anterior and posterior spiracles, the former located on the dorsal surface of prothorax, the latter posteriorly directed at the rear; strongly sclerotised mouthparts, the mandibles with its longitudinal axis at about right angles to the rest of the cephalopharyngeal skeleton and usually bearing two or more pairs of equal sized anteriorly directed teeth.

Genus Liriomyza - Adult of the genus Liriomyza have the following morphological characters: small flies, 1-3 mm in length; fronto-orbital setulae reclinate (backward pointing) (Figure 6); usually with a dark pre-scutellar area concolorous with the scutum (Figure 7) , rarely yellow; scutellum yellow in most species (Figure 7), rarely dark (Figure 8); costa extends to vein M1+2 (Figure 9, compare with Figure 10); discal cell small (Figure 9); second crossvein present (Figure 9, compare with Figure 10) in most species; stridulating organ present in males (a “scraper”, a chitinized ridge on the hind-femora, and a “file”, a line of low chitinized scales on the connecting membrane between the abdominal tergites and sternites). Morphological differences between many closely related species of Liriomyza are slight, but the minor colour differences which are detectable, are remarkably constant. However, a positive identification of many such species is only possible from examination of the male genitalia (Spencer, 1972).

Other common Australian genera of Agromyzidae and Drosophilidae that are leafminers and may be confused with Liriomyza -

The very close genera (common species C. syngenesiae) and , (of which Australia has 21 species), can be separated from Liriomyza by their proclinate (forward pointing) orbital setulae (always reclinate or occasionally upright or missing in Liriomyza), scutellum generally grey or black but occasionally slightly yellowish centrally (bright yellow in L. trifolii), and a distinct male genitalia. Phytoliriomyza species are gall-forming (on stem or leaf) internal feeders whereas Chromatomyia and Liriomyza species are typically leafminers. In Phytoliriomyza costa extending only to R4+5 whereas in Chromatomyia extending to vein M1+2 (Spencer, 1977). The drosophilid Scaptomyza flaveola, recorded as a common leafminer on cultivated brassicas in Tasmania (Osmelak, 1983) and in Victoria can be distinguished from Liriomyza by the lack of incurved lower fronto-orbital bristles, uniformly pale body colour and characteristic male genitalia (incurved lower fronto-orbital bristles present, body usually dark with scutellum yellow and characteristic male genitalia in Liriomyza).

Australian Liriomyza - All Australian species of Liriomyza should be considered, as they all could be potentially confused with L. trifolii. However, to distinguish L. trifolii from these, mostly non-pest, Australian species, the following combination of characters can be used: frons bright yellow (Figure 11), colour of head under outer and inner vertical bristles yellow (Figure 12, compare with Figure 13); colour of hind margin of eyes mostly yellow (Figures 1 & 2); third antennal segment yellow (Figure 11, compare with 14); scutellum yellow (Figure 7, compare with Figure 8), mesonotum greyish mat; femora yellow with occasional slight brownish striations (Figure 15, compare with Figures 16); wings with costa extending to apex of vein M1+2 (Figure 9, compare with Figure 10), second cross vein present (Figure 9, compare with Figure 10), length of last section of M3+4 3 to 4 times length of penultimate (Figure 9); male distiphallus with one distal bulb (Figure 17, compare with Figure 18) with a marked constriction between lower and upper halves.

Other exotic Liriomyza - For many years L. trifolii was confused with the vegetable leafminer, L. sativae (not yet found in Australia), as both have much the same distribution and host range. But they are readily distinguishable from each other: L. trifolii has both vertical bristles on yellow ground (outer vertical bristle on black ground in L. sativae), a matt greyish mesonotum (shining black in L. sativae), yellow occasional slight brownish striations on femora (bright yellow femora in L. sativae), and male distiphallus with one distal bulb with a marked constriction between lower and upper halves (one distal bulb with a slight constriction between upper and lower halves in L. sativae).

4. Comparative images of symptoms/damage/trauma caused by American serpentine leafminer

See Figures 19 & 20. Feeding punctures made by female flies are rounded and usually about 0.2 mm in diameter, and appear as white speckles on the upper leaf surface. The larvae feed mostly in the upper part of the leaf, mining through the green pallisade tissue. Mines are usually off-white, with trails of frass appearing as broken black strips along their length. The typical mines are tightly coiled and almost blotch-like.

5. Sampling technique

a. Number of specimens to be collected A large sample of specimens would be preferable. The aim is to obtain an adult male. Adult females are identifiable with certainty only to genus level; therefore males are needed to examine genitalia details to confirm species identification. b. Preferred stage to be collected Of the four life stages (egg, , and adult) only adults are identifiable to species using morphological features. Larvae and pupae are identifiable to species using electrophoretic and molecular tests only. c. How to collect Adult flies can be hand collected into glass vials or vacuum collected either with vacuum sampler, or swept from foliage with a hand net. Adult flies are normally found on the foliage. However the most practical and reliable method is the collection of leaves with mines containing pupae or mature larvae in a large jar for rearing in the laboratory for obtaining adult flies. d. How to collect plant sample if required Leaves with suspect feeding punctures or leaf mines should be picked and placed between sheets of newspaper to permit slow drying. For laboratory rearing of adult flies, mined leaves containing pupae or mature larvae can be collected in a large jar and kept in a constant temperature room for regular checking. e. How to preserve plant sample Leaves with suspect feeding punctures or leaf mines can be stored between sheets of dry newspaper. f. How to preserve leafminers Adults and larvae can be placed in 70% ethanol and stored indefinitely, although their colour fades gradually with time. Specimens required for molecular diagnostic work should be killed and preserved in 100% ethanol or frozen (- 80 C). g. How to transport leafminers Vials of ethanol should be sealed to avoid leakage and packed with cushioning material in a strong box. h. How to transport plant sample

Leaves with suspect feeding punctures or leaf mines should be mailed as a flat package between sheets of dry newspaper.

6. Diagnostic tests for American serpentine leafminer a. Morphological identification of adult flies

Key to distinguish the exotic , L. cicerina, L. huidobrensis and L. sativae from the Australian leafminer species of the genus Liriomyza 1. Scutellum largely black (e.g. Figure 8), sometimes brownish or greyish, never yellow ………………………………………………………………….……(L. helichrysi, L. primitiva, L. scaevolae and non- Liriomyza leafminers) -. Scutellum distinctly yellow, at least centrally (Figure 7).……………….…..…..…..…….2

2. Second cross-vein in wing lacking (Figure 10)…………………………….(L. caulophaga, L. chenopodii, L. lepida, L. meracula, L. oleariana, L. tenera) -. Second cross-vein in wing present (Figure 9)……………..………….………..………….3

3. Femora yellow, without dark striations (Figure 16)..……………….……….……………4

-. Femora yellow, with dark striations (Figure 15)………..………….………….……..……5

4. Male aedeagus with distiphallus with one distal bulb with slight constriction between lower and upper halves (e.g. Figure 17); [vertex of head black under outer vertical bristles and yellow under inner bristles (Figure 13); shining black mesonotum]……………...…L. sativae

-. Male aedeagus with distiphallus not as above; vertex of head black under inner and outer vertical bristles or yellow under inner bristles……………………………. L. brassicae, L. cassiniae, L. compositella, L. electa, L. languida, L. obscurata)

5. Vertex of head yellow under outer vertical bristles (Figure 12); [male distiphallus as in Figure 17]…………………………………………………………….……...... …...L. trifolii -. Vertex of head black under outer vertical bristles (Figure 13)……..…………………… 6

6. Third antennal segment yellow (Figure 11); last section of wing vein M3+4 < 2 times length of penultimate……………………………………………………………....……L. inopinata -. Third antennal segment dark, at least partially (Figure 14); [last section of wing vein M3+4 ≥ 2 times length of penultimate (Figure 7)].………………………..…………….……………7

7. Male aedeagus with single lobed distiphallus (e.g., Figure 17); scutellum bright yellow centrally, black at sides; mesonotum largely matt ……………………..…………………….L. impolita -. Male aedeagus with bilobed distiphallus (Figure 18); scutellum yellow; mesonotum shining black……………………………………………………………………..……………..….. 8

8. Entire 3rd antennal segment variably black; male distiphallus with distal lobes meeting from their rims to their bases; smaller species, wing length 1.3 – 1.5 mm; Leguminosae only..……………………………………………………….…………………..…L. cicerina -. Only distal 1/3 antennal segment dark; male distiphallus with distal lobes meeting only at their rims; larger species, wing length 1.7 – 2.2 mm; polyphagous..……..….L. huidobrensis

b. Electrophoretic identification of larvae and pupae The detailed methodology is provided in Appendix A of diagnostic protocols developed by Central Science Laboratory, UK, see following website, http://www.csl.gov.uk/science/organ/ph/diagpro/liriomyza.pdf c. Molecular diagnostic tests for larvae and pupae

Dr Sonja Scheffer of Systematic Entomology Laboratory, USDA has the capability to undertake molecular diagnostic tests for a number of Liriomyza species including L. trifolii (eg. Scheffer, 2000). In Australia Dr Tracy Bjorksten of CESAR Melbourne University has recently initiated the development of this capability, and it is intended that the Department of Primary Industries Victoria at Knoxfield Centre will be undertaking the completion of this development, as well as the operation of this diagnostic capability from about the middle of 2005.

7. Information on American serpentine leafminer

Species: Liriomyza trifolii (Burgess, 1880)

Synonyms: phaseolunata Frost, 1943; Liriomyza alliovora Frick, 1955; Liriomyza phaseolunata (Frost, 1943); Liriomyza trifolii de Meijere, 1925; Oscinis trifolii Burgess, 1880

Taxonomic position: Insecta: Diptera: Agromyzidae

Common names: American serpentine leafminer (English); leafminer (English); leafminer (English); serpentine leafminer (English) mineuse du (French) Floridaminierfliege (German) minador pequeño del frijol (Spanish)

Known hosts: Liriomyza trifolii attacks a wide range of ornamental and vegetable crops. The host range encompasses over 400 species of plants in 28 families (CABI, 2003; Reitz and Trumble, 2002). This extreme degree of polyphagy is unusual among species of Liriomyza; only five of the more than 300 species of the genus are considered to be truly polyphagous (Spencer, 1973). Hosts: Basellaceae: Basella; Caryophyllaceae: Dianthus (carnation), Gypsophila (baby's breath); Chenopodiaceae: Beta vulgaris var. saccharifera (sugarbeet), Chenopodium (goosefoot), Spinacia oleracea (spinach); Compositae: Ageratum, Ambrosia (ragweed), Artemisia (wormwoods), Aster, Baccharis, Bellis, Bidens (burmarigold), Callistephus, Carthamus, Centaurea (knapweed), Chrysanthemum (daisy), Chrysanthemum x morifolium (chrysanthemum (florists')), Dahlia, Dendranthema, Erigeron (fleabane), Eupatorium, Gaillardia, Gazania, Gerbera (Barbeton daisy), Helianthus, Lactuca sativae (lettuce), Senecio (groundsel), Sonchus (milkthistle), Tagetes, Taraxacum (dandelion), Tithonia, Tragopogon, Xanthium (cocklebur), Zinnia; Convolvulaceae: Ipomoea (morning glory); Cruciferae: Brassica rapa subsp. chinensis (Chinese ); Cucurbitaceae: Citrullus, Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita pepo (ornamental gourd); Euphorbiaceae: Ricinus; Gramineae: Avena sativa (oats), Hordeum (barleys); Iridaceae: Gladiolus hybrids (sword lily); Labiatae: Moluccella, Ocimum, Salvia; Leguminosae: Arachis hypogaea (groundnut), Cassia (sennas), Crotalaria, Glycine, Glycine max

(soyabean), Lathyrus (vetchling), Macrotyloma, Medicago (medic), Medicago sativae (lucerne), Melilotus (melilots), Phaseolus (beans), Phaseolus lunatus (lima bean), Phaseolus vulgaris (common bean), Pisum sativum (pea), Trifolium (clovers), Trifolium repens (white clover), Trigonella, Vicia (vetch), Vigna unguiculata (cowpea); Liliaceae: Allium (onions, garlic, leek, etc.), Allium cepa (onion), Allium sativum (garlic), Allium schoenoprasum (chives), Alstroemeria (Inca lily); Malvaceae: Abelmoschus esculentus (okra), Gossypium (cotton), Malva (mallow); Polemomiaceae: Phlox; Primulaceae: Primula (primrose); Rosaceae: Crataegus (hawthorns); Scrophulariaceae: Antirrhinum (snapdragon), Linaria (toadflax); Solanaceae: Capsicum annuum (bell pepper), Cestrum, Lycopersicon esculentum (), Physalis (groundcherry), Solanum melongena (aubergine), Solanum tuberosum (potato); Tropaeolaceae: Tropaeolum; Typhaceae: Typha; Umbelliferae: Apium graveolens var. dulce (bleached celery), Daucus carota (carrot); Verbenaceae: Verbena; Zygophyllaceae: Tribulus.

Distribution: Africa: Benin, Côte d'Ivoire, Egypt, Ethiopia, Guinea, Kenya, Madagascar, Mauritius, Mayotte, Nigeria, Réunion, Senegal, South Africa, Sudan, Tanzania, Tunisia, Zambia, Zimbabwe Asia: China, India, Israel, Japan, Jordan, Korea (Republic of), Lebanon, Oman, Philippines, Thailand, Turkey, Yemen Central America: Bahamas, Barbados, British Virgin Islands, Costa Rica, Cuba, Dominican Republic, Guadeloupe, Guatemala, Martinique, Netherlands Antilles, Puerti Rica, Trinidad and Tobago, Virgin Islands : Austria, Belgium, Croatia, Cyprus, France, Greece, Hungary, Italy, Malta, Netherlands, Norway, Portugal, Romania, Russian Federation, Slovenia, Spain, Switzerland, Yugoslavia North America: Bermuda, Canada, Mexico, USA Oceania: American Samoa, Federated states of Micronesia, Guam, Northern Mariana Islands, Samoa, Tonga South America: Argentina, Brazil, Chile, Colombia, Ecuador, French Guiana, Guyana, Peru, Venezuela

For further details see EPPO (2004) and CABI (2004).

Part of Plants Affected: Leaf. Adult feeding punctures and larval feeding mines occur only on leaves (Figures 19 & 20).

Features: Egg (Figure 21) Eggs of L. trifolii are 0.2-0.3 mm X 0.1-0.15 mm, off white and slightly translucent.

Larva (Figure 22) This is a legless maggot with no distinct or separate head capsule. It is transparent when newly hatched but turns to yellow-orange in later instars, and is up to 3 mm long. There are three larval stages. The two posterior spiracles each have three pores, the outer two elongate.

Pupa (Figure 23) This is oval, distinctly segmented, and slightly flattened ventrally, 1.3-2.3 mm X 0.5-0.75 mm, with variable colour, pale yellow-orange darkening to golden-brown.The two posterior spiracles are on a pronounced conical projection, and each has three distinct bulbs, two of which are elongate. Adult (Figures 1 & 2) Liriomyza trifolii is a very small species (1.0 -1.3 mm body length, and up to 1.7 mm in females) with conspicuously matt greyish mesonotum and both vertical bristles on yellow ground. All antennal segments, face and frons are bright yellow. The hind margin of the eye is largely yellow. The third antennal segment is noticeably but finely pubescent. The orbital setulae are sparse and reclinate. The mesonotum has conspicuous yellow patches at each hind-corner adjoining the scutellum, which is bright yellow. The mesopleura are predominantly yellow with a small blackish- grey patch on the lower margin, and the sternopleura are largely black with the upper margin yellow. The coxae are yellow, the femora are largely yellow but with slight, variable brownish striation, and the tibiae and tarsi are darker, brown. The abdomen is largely black but the tergites are variably yellow, particularly at the sides. The squamae are yellowish, with a dark margin and fringe. The wings are 1.3-1.7 mm in length. Male and female L. trifolii are generally similar in appearance. In the male the distiphallus is distinctly constricted apically, the neck behind adjoining and relatively long, a little shorter than the distiphallus proper (Figure 17). A detailed description of the species is found in Spencer (1973).

Biology: Life history: The rate of immature development of L. trifolii is dependent on temperature. At a uniform temperature of 28°C one generation cycle can be accomplished in 14-15 days, but at lower temperatures the time taken is progressively longer. At 16°C pupal diapause begins and winter generations of pupa will remain in the soil until warmer conditions occur again. The adult can survive temperatures down to about 12°C but does not appear to feed or lay eggs. In heated glasshouses where suitable hosts may be grown throughout the year, the breeding and development of L. trifolii will be virtually continuous. In cool glasshouses generation rates will be different throughout the seasons, with fairly rapid development during the summer and pupa remaining undeveloped in the soil during the coldest periods (Ref…) ????. L. trifolii eggs are inserted just below the leaf surface and hatch in 2-5 days according to temperature. Harris and Tate (1933) give 4-7 days at 24°C. Many eggs may be laid on a single leaf. Female L. trifolii flies puncture the leaves of the host plants causing wounds which serve as sites for feeding or oviposition. Feeding punctures of Liriomyza species are rounded and usually about 0.2 mm in diameter. They appear as white speckles on the upper leaf surface. The appearance of the punctures does not differ between species, nor can the pattern of their distribution on the leaf be used to separate species. Feeding punctures cause the destruction of a large number of cells and are clearly visible to the naked eye. About 15% of oviposition punctures made by L. trifolii contain viable eggs (Parrella et al., 1981). Male L. trifolii are unable to puncture the leaves but have been observed feeding at punctures made by females. Both male and female L. trifolii feed on dilute honey (in the laboratory) and take nectar from flowers (EPPO, 1990). The larval mine is on the upper or lower surface of the leaf, in the leaf mesophyll tissue, and is linear,

shallow, at first greenish, later whitish, winding irregularly and frequently forming a secondary blotch, and with trails of frass appearing as broken black strips along their length. Feeding punctures and leaf mines are usually the first and most obvious sign of the presence of Liriomyza. They remain intact and relatively unchanged over a period of weeks. The duration of larval development also depends on temperature and probably host plant. Several generations can occur during the year, breeding only being restricted by the temperature and the availability of fresh plant growth in suitable hosts (Spencer, 1973). L. trifolii pupation occurs outside the leaf, in the soil beneath the plant. Pupa development will vary according to season and temperature. Adult emergence occurs 7-14 days after pupation at temperatures between 20 and 30°C (Leibee, 1982). Wolfenbarger (1947) gives 24-28 days for the complete cycle, in during December-January (winter period). Peak emergence of adult L. trifolii occurs before midday (McGregor, 1914). Males usually emerge before females. Mating takes place from 24 hours after emergence and a single mating is sufficient to fertilise all a female's eggs. Adults of L. trifolii live between 15 and 30 days. On average, females live longer than males.

Control strategies: - Numerous parasitic wasps (Hymenoptera) occurring naturally, may be used for control of L. trifolii. These wasps are difficult to isolate or identify and local agricultural advisory services should be consulted about which species are available and natural in that locality, and their artificial introduction. There has been considerable work on natural enemies in relation to biological control introduction programmes. Waterhouse and Norris (1987) give a detailed list of the natural enemies of Liriomyza spp. and a summary of the results of the biological control introductions against L. trifolii. Foliar applications of the entomophagous nematode, Steinernema carpocapsae, significantly reduced adult development of L. trifolii (Harris et al., 1990). A Lucid key to the world genera of (Hymenoptera) parasites of leafmining Agromyzidae by Placido Reina and John LaSalle is available on CSIRO website.

Natural Enemy List Pathogens: Bacillus thuringiensis kurstaki [check reference ] Parasitoids: Chrysocharis ainsliei; Chrysocharis caribea attacking larvae in ; Chrysocharis oscinidis attacking larvae in USA, Hawaii (intro), Mariana Is (intro), Tonga (intro); Chrysocharis parksi; Chrysocharis punctiventris; Chrysonotomyia punctiventris attacking larvae in USA, Mexico, Hawaii (intro); Chrysonotomyia punctiventris; Closterocerus purpureus attacking larvae in Trinidad; Closterocerus trifasciatus attacking larvae in USA; Closterocerus utahensis; Cothonaspis pacifica; Dacnusa sibirica; Diaulinopsis callichroma; Diglyphus begini attacking larvae in USA, Colombia; Diglyphus intermedius attacking larvae in USA; Diglyphus isaea attacking larvae in USA, Belgium, Canary Islands, Israel; Diglyphus pulchripes; Eucoilidea fetura; Eucoilidea guamensis; Eucoilidea micromorpha; Ganaspidium hunteri attacking larvae in USA, Hawaii (intro); Ganaspidium utilis attacking larvae in Hawaii, Tonga (intro), Mariana Islands (intro); Gnaptodon pumilio; Halticoptera circulus attacking larvae in USA, Trinidad, Hawaii (intro); Hemiptarsenus varicornis attacking larvae in Western Samoa, Tonga, Reunion, Senegal; Neochrysocharis formosa attacking larvae in USA; Oenonogastra microrhopalae; Opius bruneipes; Opius dimidiatus attacking larvae in USA; Opius dissitus attacking larvae in USA, Hawaii (intro); Opius pallipes; Rhizarcha lestes; Steinernema carpocapsae attacking eggs

Predators: Dicyphus tamaninii; Orius dissitus. Nematodes: Neoaplectana feltiae.

Entry potential: Low via pulses as it attacks only leaves and is not associated with seeds. Medium via other hosts, particularly on ornamentals as it has a very broad host range. Likely to come into Australia on leaves of imported flowers, as eggs i.e. before mines have developed. Depending upon the type of treatment at port of entry, the eggs and larvae ( internal feeders) are likely to survive the treatment. Eggs and larvae are likely to be on infested leaf and pupae outside the leaf.

Establishment potential: High. This leafminer has a high reproductive rate, and development is continuous as long as conditions are favourable.

Spread potential: Medium. Because L. trifolii attacks a wide range of ornamental and vegetable crops, transportation of these plants could potentially contribute to its spread. Dispersal within the crop is by flight. These leafminers are not active fliers, and in crops showing active mining, the flies may be seen walking rapidly over the leaves with only short jerky flights to adjacent leaves.

Economic Importance: Liriomyza trifolii is a major pest of the Compositae (particularly ) worldwide, as well as causing considerable damage on other hosts in 28 families. In the EPPO region it is a major pest of a wide variety of ornamental or vegetable crops grown under glass (Lactuca, Dendranthema, Gypsophila, Dahlia) or as protected crops. Vegetable losses in the USA are considerable. For example, losses for celery were estimated at US$ 9 million in 1980 Spencer, 1982). Further details in CABI (2004).

Liriomyza trifolii is known to be vector of plant viruses (Zitter et al., 1980).

Consequences: Damage is caused by L. trifolii larvae mining into leaves and occasionally petiole. The photosynthetic ability of the plants is often greatly reduced as the chlorophyll-containing cells are destroyed. Severely infested leaves may fall, exposing plant stems to wind action, and flower buds and developing fruit to scald (Musgrave et al., 1975). The presence of unsightly larval mines and adult punctures caused by L. trifolii in the leaf palisade of ornamental plants, such as chrysanthemums, can further reduce plant value (Smith et al., 1962; Musgrave et al., 1975). In young plants and seedlings, L. trifolii mining may cause considerable delay in plant development, even leading to plant loss. The chemical control of leafminers is difficult, and the systemic effective in their control (abamectin and cyromazine) may not be needed for use against any other pests, this may cause additional environmental pollution.

Mention resistance??????

Diagnostic Protocol for the Detection of the Chickpea Leafminer, Liriomyza cicerina (Diptera: Agromyzidae)

1. Diagnostic images of chickpea leafminer

No images are available.

2. Diagnostic images of Australian species with which chickpea leafminer might be confused

See Figures 3-5.

3. Organisms in Australia with which chickpea leafminer might be confused

Family Agromyzidae - See under Liriomyza trifolii.

Genus Liriomyza - See under Liriomyza trifolii.

Other common Australian genera of Agromyzidae and Drosophilidae that are leafminers and can be confused with Liriomyza - See under Liriomyza trifolii

Australian Liriomyza - To distinguish L. cicerina from these, mostly non-pest, Australian species, the following combination of characters can be used: frons orange yellow (Figure 11), colour of head under outer and inner vertical bristles dark, at least outer vertical bristles on black (Figure 13, compare with Figure 12), colour of hind margin of eyes black (Figure 13); third antennal segment round and variably dark (Figure 14); scutellum yellow (Figure 7); femora yellow with variable black striations (Figure 15); wings with costa extending to apex of vein M1+2 (Figure 9), second cross vein present (Figure 9), length of last section of M3+4 about three times length of penultimate (Figure 9); male distiphallus with two distal bulbs (Figure 18, compare with Figure 17); and the hosts are only Leguminosae.

Other exotic Liriomyza - Liriomyza cicerina can be confused with another exotic leafminer of quarantine concern, L. huidobrensis, in morphological characters such as the femora yellow with variable black striations, dark areas on 3rd antennal segment, and the male disitiphallus with two distal bulbs, however the former can be distinguished from the latter in the following external morphological characters for initial identification - smaller size, wing length 1.3-1.5 mm (larger in L. huidobrensis, wing length 1.7-2.2 mm), entire 3rd antennal segment variably dark (in L. huidobrensis, only distal 1/3 dark), last segment of M3+4 3 times length of penultimate (in L. huidobreniss 2-2.5 times), and a very distinct distiphallus, and the hosts only in Leguminosae (in L. huidobrensis, highly polyphagous). Accurate identification can only be made by the examination of the male genitalia.

The non-Australian leafminer Chromatomyia horticola, a widely distributed and polyphagous Agromyzid, also attacks chickpea in Europe. This species can be separated from L. cicerina by its longer wings (2.2-2.6 mm), distinctively matt, ash-grey mesonotum and scutellum (e.g. Figure 8), black legs with yellow knees, distinctly proclinate (forward pointing) orbital setulae, and a very distinct male genitalia (Spencer, 1973).

4. Comparative images of symptoms/damage/trauma caused by chickpea leafminer

Unavailable.

5. Sampling technique

See under Liriomyza trifolii.

6. Diagnostic keys for chickpea leafminer b. Morphological identification of adult flies

Key to distinguish the exotic Liriomyza cicerina from the Australian species of the genus Liriomyza

See under Liriomyza trifolii.

b. Electrophoretic and molecular tests for the identification of larvae and pupae

These need to be developed as yet no tests are available for this species.

7. Information on chickpea leafminer

Species: Liriomyza cicerina (Rondani, 1875)

Synonyms: Agromyza cicerina Rondani; Liriomyza ononidis de Meijere; Liriomyza trichophthalma Hering

Taxonomic position: Insecta: Diptera: Agromyzidae

Common names: chickpea leafminer (English) mosca de los garbanzales (Spanish)

Known hosts: All host species are members of the family Leguminosae. Cicer arietinum (chickpea), C. bijugum, C. chorassnicum, C. echinospermum, C. judaicum, C. pinnatifidum, C. reticulatum, C. yamashitae, Hymenocarpus circinnatus (disk trefoil), Melilotus alba (white sweetclover), M. officinalis (yellow sweetclover), Ononis species, including O. arvensis (field restharrow), O. hircina, O. repens (common restharrow), O. spinosa (spiny restharrow). Liriomyza

cicerina occurs commonly on Ononis species in Western Europe and it seems probable that this is the original host, from which it colonised Cicer arietinum when this was introduced into Southern Europe from India in classical times.

Distribution: The species is widespread in the Palaearctic and Oriental regions. Africa: Morocco, Tunisia Asia: Syria, Turkey, Jordan, Lebanon. Only one reference confirmed the occurrence of this species in India (Naresh and Malik, 1986). Europe: Romania, Slovakia, Ukraine, Yugoslavia, Portugal, Spain England, Germany, Italy, Sweden, Denmark.

Part of Plants Affected: Leaf

Features: Egg No information specific to L. cicerina available.

Larva Posterior spiracles each with 7-9 pores, the two processes widely separated. No other information specific to L. cicerina available.

Pupa Puparium pale brownish-yellow. No other information specific to L. cicerina available.

Adult Liriomyza cicerina is a very small, variably dark species with the third antennal segment round and variably dark to almost entirely dark, first and second segments yellowish. Vertex dark, at least outer vertical bristle on black ground. The wings are 1.3-1.6 mm long. The mesonotum is moderately shining black, dark to the margin of the scutellum which is yellow, mesopleura normally black along lower and front margins, occasionally with only a small black patch on lower margin. The legs dark, femora although basically yellow largely darkened by variable black striations. The halteres are yellow. A detailed description of the species is found in Spencer (1973).

Biology: Life history: There may be two to four generations a year depending on the temperature and the availability of host plants (Lahmar and Zeouienne, 1990). Development of first generation takes around 25 days (Shevtchenko, 1937). Adult females puncture the upper surface of leaves with their ovipositor for feeding purposes, and, in some of the punctures, eggs are inserted just under

the epidermis. Feeding punctures of Liriomyza species are rounded and usually about 0.2 mm in diameter. They appear as white speckles on the upper leaf surface. The appearance of the punctures does not differ between species, nor can the pattern of their distribution on the leaf be used to separate species. The egg stage lasts for 2 or 3 days. The larval mine is on the upper or lower surface of the leaf, in the leaf mesophyll tissue, and is linear, shallow, at first greenish, later whitish, winding irregularly and frequently forming a secondary blotch, and with trails of frass appearing as broken black strips along their length. Feeding punctures and leaf mines are usually the first and most obvious sign of the presence of Liriomyza. They remain intact and relatively unchanged over a period of weeks. Pupation takes place externally, after the full-grown larva has fallen to the soil. The life cycle is completed in between 20 and 30 days, the pupal stage lasting generally from 10-20 days in the early generation. In Syria the first leafminer generation emerging from diapause appears in late March. The second generation reaches peak population in mid May. When the host chickpea plants mature the leafminers disappear and it is suspected that they survive the summer and winter as pupae in diapause.

Control strategies: Research by International Centre for Agricultural Research in the Dry Areas (ICARDA) in Syria has shown that the can be effectively controlled by insecticides but their use in farmers' fields is limited because of their high prices. Using the synthesised pyrethroid insecticide, deltamethrin, reduced the number of parasitoids, by about 70%, compared to the unsprayed check (Bouhssini et al., 2004). The environmental hazards associated with the use of insecticides have to be considered, particularly the threat imposed by the insecticides to the useful natural enemies of the leafminer. Natural enemies of chickpea leafminer should be conserved as they play a major role in regulating the insect population. The two dominant species of parasitoids in Syria, among a complex of species, are Diglyphus isaea (Eulophidae) and Opius monilicornis () (CABI, 2003; Weigand, 1990). Studies show that the level of parasitism by the larval (Opius monilicornis) reaches 70% on the third generation of the leafminer. Opius pygmaeus has been confirmed parasitising L. cicerina in England (Fischer, 1962; reported in Spencer, 1973). Possibly the same species in Spain (Spencer, 1973) was found to parasitise up to 90% of larvae of the first generation, thus effectively reducing populations later in the year (Del Canizo, 1934). Pediobius acantha (Eulophidae) has been reported from L. cicerina in Turkey (Gencer, 2004). Another control measure which has provided partial success is the screening for resistance against leafminers (Singh et al., 1997; Singh et al., 1998).

Entry potential: Low as it attacks only leaves and is not associated with seeds, entry most likely by accidental transport, adults survive off host plants only for a short period . Eggs and larvae are likely to be on infested leaf and pupae outside the leaf.

Establishment potential: Medium because this leafminer has a high reproductive rate, and development is continuous as long as conditions are favourable.

Spread potential: Low because L. cicerina attacks only Leguminosae and transportation of these plants could potentially contribute to its spread. Dispersal within the crop is by flight. These leafminers are not active fliers, and in crops showing active mining, the flies may be seen walking rapidly over the leaves with only short jerky flights to adjacent leaves.

Economic importance: Chickpea is used for both human consumption and forage plant for livestock particularly in the Mediterranean region and India (van Rheenen, 1991). In areas where chickpea is grown, Liriomyza cicerina must be considered one of the most serious pests, as it can severely affect the vitality of the plants and reduce the amount and the quality of the yield (e.g. Pastucha, 1996). Lower leaves are attacked first and often only three or four healthy leaves remain on each stem. The resulting reduction in assimilation by the plant inevitably reduces its vitality and leads to a significant loss in the harvest. On plants suffering partial attack the pods are smaller and less numerous than normal, while in cases of heavy attack, plants fail to produce any pods. Insecticide trials in Syria have shown that losses due to leafminers range from 0 to 30% seed yield. Shevtchenko (1937) found in the Ukraine up to 1852 puparia per square metrer of soil. Damage to leaves actively facilitates subsequent fungal attack, reported as being caused by Phyllosticta rabiei (= Ascochyta rabiei), ascochyta blight, in Spain (Del Canizo, 1934, 1945; reported in Spencer, 1973), and by 'Ascochyta' in the Ukraine (Shevtchenko, 1937)

Consequences: The mining activity of larvae reduces the photosynthetic capacity of the plant and heavy infestation will cause desiccation and premature fall of leaves. The environmental hazards associated with the use of insecticides to control this pest have to be considered, particularly the threat imposed by the insecticides to the useful natural enemies of the leafminer. Natural enemies of chickpea leafminer should be conserved as they play a major role in regulating the insect population. Note that the agromyzid parasitoids are relatively polyphagous and that it is likely that the local parasitoid fauna will provide substantial levels of control, provided they are not disrupted by broad-spectrum insecticides.

Some of the chemicals used in the control of leafminers may pose a minor health risk to applicators.

References:

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Canizo, J. Del (1934). Dos Agromicides perjudiciales al garbanzo. Boletin de Patologia Vegetal y Entomologia Agricola 7: 91-103.

Canizo, J. Del (1945). Notas sobre la mosca del garbanzo. Boletin de Patologia Vegetal y Entomologia Agricola 13: 15-26.

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Frick, K.E. (1955). Nearctic species in the Liriomyza pusilla complex, No. 3. L. alliovora, new name for the Iowa onion miner. Journal of Kansas Entomological Society 28: 88-92.

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Gencer, L. (2004). A study on the Chalcidoid (Hymenoptera: Chalcidoidea) parasitoids of leafminers (Diptera: Agromyzidae) in Ankara Province. Turkish Journal of Zoology 28: 119-122. http://journals.tubitak.gov.tr/zoology/issues/zoo-04-28-2/zoo-28-2-5-0211-7.pdf .

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Harris, M.A., Begley, J.W. and Warkentin, D.L. (1990). Liriomyza trifolii (Diptera: Agromyzidae) suppression with foliar applications of Steinernema carpocapsae (Rhabditida: Steinernematidae) and abamectin. Journal of Economic Entomology, 83(6): 2380-2384

Kolesik, P. and Pastucha, L. (1992). Chickpea leafminer Liriomyza cicerina (Rondani, 1875) [Diptera, Agromyzidae] – a new species of Czecho-Slovak fauna. Biologia (Bratislava), 47 (5): ….? Lahmar, M. and Zeouienne, M. (1990). Biological data and importance of damage of chickpea leaf miner (Liriomyza cicerina Rond.) in Morocco. Al-Awamia (Morocco) 72: 108-118.

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Preparedness for Specialist Emergency Diagnosis a. Definitive diagnosis of Liriomyza species requires examination of male genitalia and comparison with validated voucher specimens. Experience with identification of Liriomyza and other related Agromyzidae and the ability to interpret and relate structures, particularly those of male genitalia, should be demonstrated. b. Proposed methodology for diagnosis: 1. Examine adult specimens under dissecting microscope to assess whether or not they belong to genus Liriomyza using external morphological characters listed under section 1 above. 2. If they are Liriomyza, then key through the identification key given under section 6 above to distinguish them from the Australian species of this genus. 3. If the specimens key to L. trifolii or L. cicerina, select male specimen and carefully detach abdomen from specimen. 4. Soak abdomen in 10% KOH for several hours until it becomes soft and cleared. Wash in water. 5. Carefully dissect out the genitalia and mount in Hoyer’s media on a microscope slide. Remainder of the abdomen may also be mounted on the same slide but under a separate cover slip. 6. Cross label the slide with the data on the remainder of the specimen left in alcohol. 7. Now the slide is ready for examination. Place slide in an oven (incubator) at 40 - 45 C for drying for a few days before storing in the collection. c. Commitment Specimens from Australia suspected to be L. trifolii or L. cicerina (or other exotic species such as L. huidobrensis and L. sativae) can be sent to Dr Mali Malipatil, Department of Primary Industries Victoria, PMB 15, Ferntree Gully Delivery Centre, Victoria 3156 for confirmation. No charge will be made for a determination of whether a particular specimen is or is not L. trifolii or L. cicerina. Further identification of a specimen that is not L. trifolii or L. cicerina may attract a charge. The Victorian Agricultural Insect Collection of the DPI Victoria has acquired adult specimens of L. trifolii and is in the process of acquiring L. cicerina specimens for comparison and has copies of the major taxonomic literature enabling positive identification of both these species to be made. A positive species identification of larvae and pupae can only be done by molecular tests (yet to be developed for L. cicerina). The specimens needed to be preserved in 100% ethanol or frozen and forwarded to Dr Malipatil at the above address. He will organise for the testing through Professor Ary Hoffmann of CESAR, Melbourne University.

Positive identification of L. trifolii or L. cicerina (or other exotic species such as L. huidobrensis and L. sativae) from Australia will be immediately communicated to appropriate authorities within DAFF including details of : . Overseas source of sample; . Details of associated plant material;

. Estimated size of infestation; . Reported damage, if any; . Implications of the incursion; . Reference to this protocol for images and description of the pest; . Known distribution outside Australia; . Estimate of potential distribution within Australia; . Level of confidence in identity; . Date of confirmed identification.