Liriomyza trifolii (American serpentine leafminer)

L. trifolii is a leaf-mining insect, commonly known as the serpentine leafminer. It is highly polyphagous and has been recorded from 25 families. As a major pest of ornamental and vegetable crops, including beans (phaseolus), Capsicum, carnations, celery, chrysanthemums (Dendranthenum, the commercial 'Mum'), clover, Cucumis, Gerbera, Gypsophila, lettuces, lucerne, potatoes, Senecio hybridus and tomatoes it has had important biological and economic impacts across a number of countries, including North America, Africa and Europe.

Liriomyza trifolii is one of the truly polyphagous agromyzids and has been recorded in 25 families (Spencer, 1990). It was first described as Oscinis trifolii (Burgess in Comstock, 1880) in the family Chloropidae from flies attacking the leaves of Trifolium repens (white clover) in Indiana, USA. Later, it was transferred to the family Agromyzidae in the genus Agromyza by Coquillet (1898), then to Liriomyza by de Meijere (1925). Spencer, 1973 synonymized Liriomyza alliovora Frick, 1955, breeding in Allium (onions) in Iowa, USA, and in Spencer, 1986, Agromyza phaseolunata Frost (1943, as Liriomyza) attacking Phaseolus lunatus (lima beans) in New Jersey, USA with L. trifolii.
 

Descriptions of L. trifolii refer to fresh materials. Dry specimens may be distorted due to the manner in which they have been preserved. Also, the age of the specimen, when killed, will have some effect on its preservation characteristics.

For accurate identification, examination of the leaf mine and all stages of development are crucial.

Egg

L. trifolii eggs are 0.2-0.3 mm x 0.1-0.15 mm, off white and slightly translucent.

Larva

This is a legless maggot with no separate head capsule, transparent when newly hatched but colouring up to a yellow-orange in later instars and is up to 3 mm long. L. trifolii larvae and puparia have a pair of posterior spiracles terminating in three cone-like appendages. Spencer (1973) describes distinguishing features of the larvae. Petitt (1990) describes a method of identifying the different instars of the larvae of L. sativae, which can be adapted for use with the other Liriomyza species, including L. trifolii.

Puparium

This is oval and slightly flattened ventrally, 1.3-2.3 x 0.5-0.75 mm with variable colour, pale yellow-orange, darkening to golden-brown. The puparium has posterior spiracles on a pronounced conical projection, each with three distinct bulbs, two of which are elongate. Pupariation occurs outside the leaf, in the soil beneath the plant.

Menken and Ulenberg (1986) describe a method of distinguishing L. trifolii from L. bryoniae, L. huidobrensis, and L. sativae using allozyme variation patterns as revealed by gel electrophoresis.

Adult

L. trifolii is very small: 1-1.3 mm body length, up to 1.7 mm in female with wings 1.3-1.7 mm. The mesonotum is grey-black with a yellow blotch at the hind-corners. The scutellum is bright yellow; the face, frons and third antennal segment are bright yellow. Male and female L. trifolii are generally similar in appearance.

L. trifolii are not very 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.

Head
The frons, which projects very slightly above the eye, is just less than 1.5 times the width of the eye (viewed from above). There are two equal ors and two ori (the lower one weaker). Orbital setulae are sparse and reclinate. The jowls are deep (almost 0.33 times the height of the eye at the rear); the cheeks form a distinct ring below the eye. The third antennal segment is small, round and noticeably pubescent, but not excessively so (vte and vti are both on a yellow ground).

Mesonotum
Acrostical bristles occur irregularly in 3-4 rows at the front, reducing to two rows behind. There is a conspicuous yellow patch at each hind-corner. The pleura are yellow; the meso- and sterno-pleura have variable black markings.

Wing
Length 1.3 -1.7 mm, discal cell small. The last section is M<(sub)3+4> from 3-4 times the length of the penultimate one.

Genitalia
The shape of the distiphallus is fairly distinctive but could be mis-identified for L. sativae. Identification using the male genitalia should only be undertaken by specialists.

Colour
The head (including the antenna and face) is bright yellow. The hind margin of the eye is largely yellow, vte and vti always on yellow ground.

The mesopleura is predominantly yellow, with a variable dark area, from a slim grey bar along the base to extensive darkening reaching higher up the front margin than the back margin. The sternopleura is largely filled by a black triangle, but always with bright yellow above.

The femora and coxa are bright yellow, with the tibia and tarsi darker; brownish-yellow on the fore-legs, brownish-black on the hind legs. 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.

Although individual specimens may vary considerably in colour, the basic pattern is consistent.

L. trifolii has not yet been reported from many countries where it is actually present. It is generally recognized that all the countries bordering the Mediterranean have L. trifolii in varying degrees and that it occurs in all mainland states of the USA. L. trifolii has been recorded from the Juan Fernandez Islands (an offshore territory of Chile; Martinez and Etienne, 2002; EPPO, 2009). See also CABI/EPPO (1998, No. 96). L. trifolii is apparently unable to overwinter in the open in the north European EPPO countries. However,  the current regulations to prevent entry and spread in non-Mediterranean areas were found to be only partially effective, as interceptions are still being reported (EFSA, 2012).

The record for Argentina has been changed to 'Absent, unreliable record' as Martinez and Etienne (2002) and EPPO (2006) are based on Burgess (in Comstock, 1880 (1879)) and there have been no other reports of the pest in Argentina. L. trifolii is a quarantine pest for Argentina (SENASA, personal communication, 2008).

L. trifolii is listed as an A2 quarantine pest by EPPO (OEPP/EPPO, 1984). It is one of the most important recent introductions to 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 in the EPPO region. It could also cause damage to these crops grown in the open in the warmer parts of the EPPO region. It is widely distributed in the region and the success of eradication programmes which have been conducted cannot be confirmed.

The host range of L. trifolii includes over 400 species of plants in 28 families including both ornamental crops (Bogran, 2006) and vegetables (Cheri, 2012). The main host families and species include: Apiaceae (A. graveolens); Asteraceae (Aster spp., Chrysanthemum spp., Gerbera spp., Dahlia spp., Ixeris stolonifera, Lactuca sativa, Lactuca spp., Zinnia spp.); Brassicaceae (Brassica spp.); Caryophyllaceae (Gypsophila spp.); Chenopodiaceae (Spinacia oleracea, Beta vulgaris); Cucurbitaceae (Cucumis spp., Cucurbita spp.); Fabaceae (Glycine max, Medicago sativa, Phaseolus vulgaris, Pisum sativum, Pisum spp., Trifolium spp., Vicia faba); Liliaceae (A. cepa, Allium sativum) and Solanaceae (Capsicum annuum, Capsicum frutescens, Petunia spp., Solanum lycopersicum, Solanum spp.) (EFSA, 2012).

It is now considered to be the most important pest of cowpea (Vigna uniguilata), towel gourd (Luffa cylindrica), cucumber (Cucumis sativus) and many other vegetable crops in southern China (Gao, 2014). In Europe, L. trifolii is a major pest of lettuce, beans, cucumber and celery, Capsicum sp., carnations, clover, Gerbera sp., Gypsophila sp., lucerne, Senecio hybridus, potatoes and tomatoes (EFSA, 2012). It is now a major pest of the Compositae worldwide, particularly chrysanthemums (including Dendranthenum, the commercial 'Mum') in North America, Colombia, and elsewhere. It also causes severe damage to different open field crops, such as chili peppers in Mexico.

L. trifolii feeding punctures appear as white speckles between 0.13 and 0.15 mm in diameter. Oviposition punctures are usually smaller (0.05 mm) and are more uniformly round.

L. trifolii leaf mines can vary in form with the host plant, but when adequate leaf area is available they are usually long, linear, narrow and not greatly widening towards the end. They are usually greenish white.

In very small leaves the limited area for feeding results in the formation of a secondary blotch at the end of the mine, before pupariation. In Kenya, Spencer (1985) notes the growth of many L. trifolii from mines which began with a conspicuous spiral. This is not a characteristic associated with L. trifolii on other continents.

The frass is distinctive in being deposited in black strips alternately at either side of the mine (like L. sativae), but becomes more granular towards the end of the mine (unlike L. sativae) (Spencer, 1973).

Fungal destruction of the leaf may also occur as a result of infection introduced by L. trifolii from other sources during breeding activity. Wilt may occur, especially in seedlings.

Liriomyza species, in general, may be recognized by their black (sometimes brilliantly black) and yellow colouring. Particularly, the scutellum is usually yellow and distinctive.

Several pests in this genus are similar and may be mistaken for each other on quick examination. These are L. sativae (shining black mesonotum without yellow at the hindquarters, vte always and vti usually on black ground, origins probably in South America); L. huidobrensis (which has a larger discal cell, origins in South America); L. trifolii (origins probably Caribbean/Florida); L. brassicae (origins probably South America/Caribbean); L. bryoniae (origins in Europe); L. congesta (Origins in Europe/western Asia); L. strigata (origins in Europe).

The spread of Liriomyza species through international commerce and the similarities between the seven species means that identification of individual infestations must be confirmed by specialists (Spencer, 1973).

L. trifolii'sdevelopment is strictly connected with temperature. Consequently, 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 puparial diapause begins and winter generations of puparia 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 puparia remaining undeveloped in the soil during the coldest periods.

In the moderate and variable temperatures of open-field cultivation there will be only a few (perhaps three) generations produced throughout the growing season because of the longer time required to complete each cycle (Süss et al., 1984).

Egg

L. trifolii eggs are inserted just below the leaf surface. Eggs 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.

Larva

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).

Puparium

L. trifolii pupariation occurs outside the leaf, in the soil beneath the plant. Puparial development will vary according to season and temperature. Adult emergence occurs 7-14 days after pupariation at temperatures between 20 and 30°C (Leibee, 1982). Wolfenbarger (1947) gives 24-28 days for the complete cycle, in Florida during December-January (winter period).

Adult

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 fertilize all a female's eggs.

Female L. trifolii flies puncture the leaves of the host plants causing wounds which serve as sites for feeding or oviposition. 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 (OEPP/EPPO, 1990).

In the southern USA, the L. trifolii life cycle is probably continuous throughout the year. There is a noticeable first generation which reaches a peak in April (Spencer, 1973). In southern Florida, L. trifolii has two or three generations followed by a number of incomplete, overlapping generations (Spencer, 1973).

On celery L. trifolii completes its life cycle (oviposition to adult emergence) in 12 days at 35°C, 26 days at 20°C, and 54 days at 15°C (Leibee, 1982). On chrysanthemums the life-cycle is completed in 24 days at 20°C but on Vigna sinensis and Phaseolus lunatus it takes only 20 days at this temperature (Poe, 1981).

Adults of L. trifolii live between 15 and 30 days. On average, females live longer than males.

Both male and female L. trifolii may act as vectors for disease by transference during feeding or egg laying, but are not inherent carriers of disease.

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).

L. trifolii is an economically important key pest of both ornamental crops (Bogran, 2006) and vegetables (Cheri, 2012).

In Kenya, chrysanthemums were grown commercially before 1976, but L. trifolii was thought to have been introduced in contaminated cuttings from Florida (USA) in 1976, at a large propagating nursery at Masongaleni. By 1979 the nursery was closed, but the establishment of the pest in local wild hosts, and the dissemination of cuttings from the nursery to other parts of the country as well as abroad, has added L. trifolii to the other pests of East Africa. It has caused considerable crop losses and loss of overseas markets due to quarantine requirements (IPPC Secretariat, 2005).

Vegetable losses in the USA are also considerable. For example, losses for celery were estimated at US$ 9 million in 1980 (Spencer, 1982). It was noted, however, that damage to celery during the first 2 months of the 3-month growing season was insignificant and largely cosmetic, whereas considerable yield loss resulted from pest presence during the final month (Foster et al., 1988). 1.5 million larval mines per hectare were recorded from onions in Iowa (Harris et al., 1933).

Damage is caused by L. trifolii larvae mining into leaves and 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 level of damage depends on many factors, including climate suitability, host resistance, crop distribution, growing conditions, control methods in place and the degree of infestation (EFSA, 2012).

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

L. trifolii are small black and yellow flies which may be detected flying closely around host plants or moving erratically and rapidly upon the leaf surfaces. Inspection of the leaf surface will reveal punctures of the epidermis and the obvious greenish-white mines with linear grains of frass along their length. For accurate identification, examination of the leaf mine and all stages of development are crucial.

L. trifolii larvae will be found feeding at the end of the mine, or the mine will end with a small convex slit in the epidermis where the larva has left the mine to pupariate on the ground. Sometimes the puparium may be found adhering to the leaf surface, although in most cases the fully-fed larva will have found its way to the ground beneath the plant to pupariate. This is especially true in hot, dry conditions where the larva/puparia would quickly desiccate if exposed on the leaf surface. Empty puparial cases are split at the anterior end, but the head capsule is not usually separated from the rest of the case.

Mined leaves should be collected into polythene bags and transferred to a press as soon as possible. Leaves containing larvae intended for breeding should be collected into individual polythene bags, which on return to the laboratory should be slightly over-pressurized by blowing into them before sealing the end. Blowing up the bag by mouth and sealing it adds valuable carbon dioxide to the moist air mix. Constant attention is required to ensure that puparia are transferred to individual tubes until the fly emerges. If the plant material begins rotting, good material with feeding larvae must be removed to more sanitary conditions.

When puparia are observed they can be very carefully removed to tubes containing a layer of fine sand, or a small strip of blotting paper or filter paper. This should be kept damp (never wet) until the adult emerges.

On emergence, the fly should be kept for at least 24 hours to harden up. Do not allow condensation to come into contact with the fly, or it will stick to the water film and be damaged.

Field collection of the adult L. trifolii is done by netting. The use of sticky traps, especially yellow ones, placed near host plants is a very effective method of collection and estimation of infestation.

If the puparial stage is collected from the soil, care must be taken not to damage the puparial skin or death will almost certainly follow. The pupae should be stored in glass tubes on a layer of clean sand or, better still, thick filter paper. The tube must have high humidity, but be free of condensation.

When the fly emerges, it must be allowed to harden for 24 hours before killing for identification purposes. Ensure that the tube has no condensation present.

Newly emerged adult L. trifolii are generally softer than specimens aged for several days and may crinkle as drying proceeds, especially the head. The ptilinal sac may still protrude from the suture between the frons and face obliterating some important characteristics. Adults should be dried slowly in the dark in a sealed receptacle over blotting paper. If preserving wet is preferred, the live specimen should be dropped into 20-40% alcohol, and transferred to 70-90% alcohol after 2 days.