Abstract

Since 1996, as part of a cooperative ARS/CIRAD Plutella biocontrol program, 115 Plutella populations have been collected in 32 countries from cole crops and cruciferous weeds. Twenty-seven primary hymenopterous parasitoid species were found, especially Diadegma spp., Cotesia plutellae and Oomyzus sokolowskii. A number of fungal pathogens, isolates of Paecilomyces fumosoroseus, Paecilomyces sp., Metarhizium sp. and Beauveria sp. were found in Australia, Benin, Romania and Georgia respectively. Biological, biochemical and genetic differences have been shown in DBM and their natural enemy populations from different geographic origins. Marked differences have been found in the behaviour of C. plutellae and O. sokolowskii towards different populations of DBM and some inter population crossings resulted in failure or only male progeny. Metarhizium from Romania and Paecilomyces from Australia killed 70–95% of DBM larvae exposed to them; other pathogens were less effective. Biocontrol of DBM is required in the south western USA where another pest, Bemisia tabaci biotype B, present in the same habitat, is managed by biocontrol based means. For successful biocontrol of DBM it may be necessary to evaluate and select natural enemies based on their association with the target DBM population.

Keywords

Biocontrol, Diadegma, Cotesia, pathogens

Introduction

Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) the diamondback moth (DBM) is a widely distributed and damaging pest of crucifer crops, resistant to many pesticides and Bacillus thuringiensis var. kurstaki and causes about $1 billion losses annually (Talekar & Shelton 1993). More than 90 species of insect parasitoids (Ichneumonidae, Braconidae and Eulophidae) are recorded, but less than ten have biocontrol potential (Noyes 1994). More than ten species of predators are known (Coccinellidae, Syrphidae and Neuroptera) (Alam 1992). Nine species of fungal pathogens: Zoophthora radicans, Beauveria bassiana, Paecilomyces fumosoroseus, Pandora blunckii, Hirsutella sp., Erynia sp., Conidiobolus sp., Scopulariopsis sp. and Metarhizium sp. (Mercadier pers. comm.) and one species of nematode: Steinernema carpocapsae (Baur et al. 1995), have been recorded worldwide. It is generally agreed that larval and pupal parasitoids such as Diadegma insulare (Cresson), D. semiclausum Hellén, Diadromus collaris (Gravenhorst) (Ichneumonidae), Cotesia plutellae (Kurdjumov) (Braconidae) and Oomyzus sokolowskii (Kurdjumov) (Eulophidae) have the greatest control potential and egg parasitoids contribute little to natural control (Talekar & Shelton 1993).

DBM is one of the key pests in crucifer crops in the USA and any attempt at its control needs to take into account the control of other pests in the crop such as B. tabaci biotype B. Bemisia and DBM are major, region-wide pests, often occurring together in the southern USA where crucifers are an important component of the agricultural system. Both pests exhibit strong invasive potential, resistance to pesticides and, in the case of DBM, resistance to Bacillus thuringiensis. In addition, Bemisia has a wide host plant range which makes it a very serious threat to a broad range of crops. Both have spread widely due to the intervention of man in moving infested cuttings and seedlings from one part of the country to another. In the USA, losses due to Bemisia were $500–750 million/annum and to diamondback moth sporadically $100s of millions/annum. The ARS European Biological Control Laboratory, Montpellier, France, in cooperation with the Entotrop laboratory of CIRAD, Montpellier, Rhodes University, South Africa and AVRDC Taiwan, in association with ICIPE, Kenya, began foreign exploration for natural enemies of DBM in 1996. One of the aims of this cooperative work is to discover and evaluate natural enemies for biological control of Plutella xylostella in the south-western USA where B. tabaci biotype B, which is currently successfully managed by bio control based means, is also present in the same habitat. Related goals are to study the interactions of the pests and their natural enemies in the field and laboratory and contribute to the elucidation of pest and natural enemy biotypes.

Material and methods

Collections were made in many countries often in collaboration with local entomologists. The insects were brought to Montpellier under French Agriculture Department and often country of origin permits, reared through one generation at CIRAD-AMIS and identified using the CIRAD reference collection. Plutella populations were conserved in liquid nitrogen for later biotyping. Diseased insects were collected in the field, isolated in Montpellier, identified and deposited in the EBCL and ARS Ithaca NY entomopathogenic fungi collections. Parasitoids were reared through one generation and either conserved or used in experiments.

(a) DBM rearing

The population of DBM used in the experiments originated from Cotonou (Benin). Parasitoids and DBM rearing took place on Brassica oleracea L. (cv. Château Renard) in a controlled temperature room under the following conditions, temperature: 26 ± 1°C; relative humidity: 70% ± 5%; photoperiod: 12 L:12 D.

(b) Crossing experiments using O. sokolowskii

Populations of O. sokolowskii originated from DBM larvae collected at Natitingou Benin (B), Cluj in Romania (R) and near Lahore in Pakistan (P). Rearing took place under the same conditions described previously. Thirty females and six males of each O. sokolowskii population were successively introduced into separate boxes (diameter 8 cm x 5 cm high) containing 15 P. xylostella IV larvae. These conditions were maintained for 24 h, after which the larvae were withdrawn and fed until pupation.

Pupae of O. sokolowskii, recovered from DBM pupae parasitised 15 days previously, were put individually into Petri dishes. At emergence, a female from one population was put together for 24 h with a male from another population. A IV instar larva was added then withdrawn after 24 h and fed until pupation. Each of the six possible crossings was repeated five times. On emergence of the F₁, the egg-adult duration and sex ratios (number of males to females) were noted. Adult F₁ were kept for 24 hours in their respective Petri dishes, before the addition of five IV DBM larvae for a further 24 hours before withdrawal. After emergence of F₂ from those larvae, egg-adult duration and sex ratios were noted. The letters denote the geographic origins of the female (first) and the male.

(c) Oviposition behaviour in the presence of variable numbers of females

Every day, a variable number of IV larvae was exposed to parasitism for 24 h. At the end of this time larvae were withdrawn, placed in individual Petri dishes and fed until their pupation. The three tests carried out were repeated six times. Test 1/1 was (1 female Oomyzus/1 DBM larva), test 1/10 (1 female Oomyzus/10 DBM larvae) and test 10/10 (10 female Oomyzus/10 DBM larvae). The information noted was, the number of parasitised larvae per female, the number of adult parasitoids emerged per pupa, the progeny of a female, the sex ratio of the progeny, the length of the life history and 50% of the progeny observed.

(d) Morphometric study (Ratio of antennal length: body length).

The antennal lengths and body lengths of eighty individuals of each sex from each population were measured.

(e) Crossing of Cotesia plutellae populations

All Cotesia populations were reared under the same conditions, 25°C, 12h/12h photoperiod and 75% Relative Humidity. The Cotesia populations were from South Africa, Benin, Martinique, Réunion and Taiwan and were collected where no exotic Cotesia have been released. Control crossings were made between males and females from the same source to confirm compatibility and the appearance of females from F₂ crossings (C. plutellae is haploid and the presence of males only, indicates infertility). In addition, incompatibility may only be recorded in the F₂ because sterile females may be produced in the F₁ stage. The partners of each pair come from different populations and were chosen at random. The same procedure was used in the F₂ pairings. Ten replicates per combination were made. Ten all-Martinique pairs were used as a control.

(f) Genetics

Reported elsewhere in these proceedings (Pichon 2003).

(g) Pathology

Aerial conidia from three strains of fungal pathogens isolated from Plutella were produced in Petri dishes of saborau/dextrose/yeast agar. Conidia were sprayed at two concentrations onto 20 II instar Plutella larvae from each geographic strain, replicated four times/strain.

Results

The results of current exploration are presented below (Table 1).

Table 1. Number of primary DBM parasitoids by region

Area	Ichneumonidae	Braconidae	Chalcididae	Eulophidae	Total
Africa	5	3	1	1	10
N America	1	1	0	0	2
S America	2	2	2	0	6
Asia	3	2	0	1	6
Australia	0	0	0	1	1
Caribbean	1	2	0	1	4
Europe	5	3	0	2	10
Indian Ocean	2	1	0	2	5
Total species	12	7	3	4	27

Twenty seven primary parasitoid species were collected (Appendix 1), Diadegma species being the most common, with D. semiclausum Hellén present in all samples collected from the Palearctic except North Africa. Other Diadegma spp. were found in Réunion, Brazil and the Dominican Republic. Diadromus collaris (Gravenhorst) was collected in France. The most common Braconidae species was Cotesia plutellae (Kurdjumov). Dolichogenidea litae (Nixon) was found locally (North and West Africa) and Dolichogenidae sp. commonly in Brazil (Brasilia). The genus Microplitis is present in temperate regions: M. mediator (Haliday) in Romania and M. plutellae Muesebeck in USA. The eulophid, Oomyzus sokolowskii (Kurdjumov), which acts mostly as a primary parasitoid (Talekar 1997), was widespread and found in Africa, Asia, Australia and Europe (Table 2).

Table 2. DBM parasitoids (selected countries)

Country	Ichneumonidae	Braconidae	Chalcididae	Eulophidae
France	3*-***	2**	0	1***
Romania	3*-***	2*-***	0	2*-***
Réunion	2***	2*-**	0	3*-**
Brazil	1***	2*-***	1*	1*
Benin	0	1***	1*	1***
Ethiopia	2***	0	0	0
S. Africa	3*-***	2*-**	0	1***
Australia	0	0	0	1**
Pakistan	0	0	0	1***
Uzbekistan	2***	1***	0	0

Common***, Local**, Scarce*

(h) Crossings between O. sokolowskii populations

Those from Romania and Pakistan produced either a higher number of males: female or no progeny, all other crossings produced similar sex ratios and viable progeny (Table 3).

Table 3. Results of crossing different populations of O. sokolowskii, n = 5

female x male	F1		F2
	Egg-emergence (d)	sex ratio (♂:♀)	Egg-emergence (d)	sex ratio (♂-♀)
B x P	21.8 b	1:7 bc	22.2 a	1:6 b
P x B	20.0 b	1:8 bc	20.0 b	1:7 bc
B x R	23.3 b	1:13 c	21.1 ab	1:10 c
R x B	21.3 b	1:6 b	21.5 ab	1:7 bc
P x R	25.3 a	1:2 a	22.5 a	1:3 a
R x P	*	*	*	*
B x B	22.0	1:5
R x R	21.3	1:5
P x P	23.0	1:6

B: Benin; R: Romania; P: Pakistan; *: no progeny obtained; a, b, c: in a column, significantly different (ANOVA and Newman-Keuls test, at 5 % level)

(i) Oviposition behaviour

Females from the Pakistani and Romanian populations parasitised the same number of larvae regardless of numbers of hosts and number of O. sokolowskii females present during oviposition; Benin females by contrast parasitised very few larvae.

(j) Morphometric study (C. plutellae)

The ratio antennal length: body length variable in females was significantly different between two groups of populations; South Africa-Martinique and Taiwan-Benin-Réunion. The ratio was > 1 in the South Africa-Martinique group.

(k) Crossing of Cotesia plutellae

Two groups of populations were separated out (Figure 1). South Africa-Martinique and Taiwan- Benin-Réunion. They were entirely compatible with each other inside a group, but not with the second group and vice versa.

Figure 1. Results of crossing 5 populations of Cotesia plutellae represented in tree form (DARwin 3.5).

(l) Pathology

Even at the low concentrations used, M. anisopliae from Romania and P. fumosoroseus from Australia killed between 70–96% of larvae treated (Table 4).

Table 4. Percentage mortality of Plutella xylostella larvae from different geographic origins when treated with Hyphomycetes

Origins of Plutella xylostella	Metarhizium anisopliae x 107	Paecilomyces fumosoroseus x 107	Beauveria bassiana x 107
Languedoc	95	71	29
Martinique	90	79	28
South Africa	96	39	32
Benin	80	74	22

Metarhizium anisopliae: Voronetz, Romania.
Paecilomyces fumosoroseus, Atherton Tableland, Queensland, Australia.
Beauveria bassiana: Renée, Republic of Georgia.

Discussion

Both Bemisia and Plutella are introduced pests into the same crop habitat in the south western US and are vulnerable to classical biocontrol. In the case of Bemisia, cole crops act as reservoir plants for overwintering populations and sustain considerable direct damage. After the appearance of Bemisia biotype B in the early 1990s, several visible disorders of Cruciferous crops appeared at the same time in California and southern Texas (Perring et al. 1991, Elsey & Farnham 1994). Successful biocontrol based management of Bemisia in the southern USA has been achieved in 5–8 years using 3 hymenopterous parasitoids chosen after rigorous selection from 36 species/strains of natural enemies (Kirk et al. 2001).

Foreign exploration for natural enemies of Plutella and the resulting biocontrol organisms discovered, have driven the programme in Montpellier through taxonomy, genetic characterisation, to evaluation. In the past Plutella natural enemies have been collected and released without knowledge of possible biological and genetic variability within Plutella itself and its natural enemies which may have determined the outcome of introductions.

The main taxonomic problems concern the Diadegma (Fitton & Walker 1992). Noyes (1994) has shown that up to 75% of host-parasitoid records are misleading because they are based on misidentifications either of the parasitoid or of the host. They also include wrong host-parasitoid associations. The use of RAPDs PCR technology would elucidate the species and strains present.

Although Cotesia plutellae is recorded from a number of hosts, Cameron et al. (1997), basing their conclusion on laboratory tests, defined this braconid as a narrowly oligotrophic parasitoid. This situation is probably the same for all the key parasitoids of Plutella. This is an important point worthy of reflection as the indirect ecological effects of biological control are increasingly being scrutinised. Specificity of DBM parasitoids, or the lack of it, needs to be shown before releases are made. The Mediterranean region is one of the presumed centres of origin of DBM (Harcourt 1954) and crucifers (Tsunoda 1980) and the maximum diversity of the parasitoid complex may therefore be expected in this region. However we have not found an exceptionally rich natural enemy fauna there. Noteworthy were two new Diadegma species found in Ethiopia (which did not attack French DBM populations). South Africa merits special attention also as Kfir (1997) has described a little known, but richly diverse fauna of efficient parasitoids. However Smith (pers. comm., 2001) did not find an exceptional natural enemy fauna in the Eastern Cape, where Plutella is not abundant. We did not find an exceptional natural enemy fauna in Romania where Mustata (1992) had recorded a very rich fauna. However we did find many species in great abundance and this fact combined with local cultivation practices and the lack of extensive chemical control made Romania an excellent source of DBM natural enemies.

Species complexes and diverse strains may hide the full potential of parasitoids for biological control based area wide IPM. For example, the behaviour and biology of Pakistani and Romanian O. sokolowskii show good potential for their use in biological control despite reduced progeny when large numbers of parasitoids are present; by contrast, in the presence of numerous females, parasitism by Benin females is strongly decreased. In addition to these behavioural differences, the Pakistan/Romania O. sokolowskii are incompatible, suggesting that they are different strains of the same species. Equally, five populations of Cotesia plutellae divided into two compatible groups suggesting the presence of different strains. The use of RAPDs PCR technology will elucidate the species and strains of DBM and natural enemies present. In addition, strains of DBM may be associated with specific strains of parasitoids.

The results of preliminary phylogenetic and isoenzyme studies show strong polymorphism between DBM populations (Pichon, pers. comm.).

EBCL has about 1000 fungal pathogen isolates collected from more than 60 countries from a number of insect sources. The Metarhizium anisoplae collected from Plutella in eastern Romania was particularly efficacious and its potential for use in biocontrol of DBM is high. Conditions at the time of collection were ideal for fungal pathogens with heavy rain showers and 25–30°C.

In conclusion, the selection and release of natural enemies for biological control based IPM of DBM should take into account specificity of parasitoids to DBM, natural enemy/DBM population associations to ensure successful establishment and, in the south-western USA, the successfully implemented biocontrol based pest management program against Bemisia in the same habitats as DBM (Kirk et al. 2001).

References

Appendix 1. Parasitoid complex of DBM

*** common species; ** fairly common species; * rare species
Trichogrammatidae
Trichogramma sp. (in course of study)	Réunion
Ichneumonidae
Diadegma insulare (Cresson)	Canada*, USA*, Dominican Rep.*
Diadegma semiclausum Hellén	Bulgaria***, France***, Romania***, Greece***, Uzbekistan***, Georgia***, Spain***, Italy***
Diadegma leontiniae (Brethes)	Brazil***
Diadegma mollipa (Holmgren)	S. Africa***, Réunion***
Diadromus collaris (Gravenhorst)	France***, S. Africa*, Turkey***, Bulgaria***, Uzbekistan, Greece**
Diadromus subtilis (Grav.)	Georgia***
Diadegma 2 spp.	Ethiopia
Diadromus sp.	France*
Diadromus subtilis (Grav.)	Georgia***
Hyposoter sp.	Romania**
Itoplectis sp.	Hungary*, Romania*, Réunion*, S. Africa*
Braconidae
Cotesia plutellae (Kurdjumov)	France**, Benin***, Réunion**, Hong Kong***, Guadeloupe**, Martinique***, Brazil*, Japan*, Uzbekistan***, Bulgaria***, Senegal*, Turkey***, S. Africa**, Italy***, Vietnam*, Canada**, Greece*
Apanteles litae (Nixon)	Tunisia**, Senegal**, Ivory Coast***, Benin**, Mali**
Apanteles sp.	France*, Austria*, Romania**
Apanteles piceotrichosus (Blanchard)	Brazil***
Apanteles eriophyes (Nixon)	S. Africa*
Glyptapanteles sp.	Martinique*, Réunion*
Microplitis mediator (Haliday)	Romania**
Microplitis Plutellae Muesebeck	Canada,* USA*, Taiwan**, Laos,* Cambodia*
Chalcididae
Brachymeria sp.	Senegal*, Benin*
Conura sp.	Brazil*
C. pseudofulvovariegata (Becker)	Martinique*, Brazil*
Eulophidae
Euplectrus sp.	Romania*
Tetrastichus howardi (Olliff)	Réunion*
Tetrastichus sp.	Réunion*
Oomyzus sokolowskii (Kurdjumov) (Occasionally facultative hyperparasitoid)	France***, Romania***, Senegal***, Benin***, Réunion*, India***, Brazil**, Guadeloupe***, Martinique***, Pakistan**, Bulgaria*, Mali**, Turkey***, S. Africa***, Italy***, Greece*