No inbreeding depression in laboratory‐reared individuals of the parasitoid wasp Allotropa burrelli

Inbreeding depression is a major concern in almost all human activities relating to plant and animal breeding. The biological control of pests with natural enemies is no exception, because populations of biocontrol agents experience a series of bottlenecks during importation, rearing, and introduction. A classical biological control program for the Comstock mealybug Pseudococcus comstocki (Hemiptera: Pseudococcidae) was initiated in France in 2008, based on the introduction of an exotic parasitoid, Allotropa burrelli Mues. (Hymenoptera: Platygastridae), a haplodiploid parasitoid imported from Japan. We evaluated the sensitivity of A. burrelli to inbreeding, to optimize rearing and release strategies. We compared several morphological and life‐history traits between the offspring of siblings and the offspring of unrelated parents. We took into account the low level of genetic variability due to the relatively small size of laboratory‐reared populations by contrasting two types of pedigree: one for individuals from a strain founded from a single field population, and the other generated by hybridizing individuals from two strains founded from two highly differentiated populations. Despite this careful design, we obtained no evidence for a negative impact of inbreeding on laboratory‐reared A. burrelli. We discussed the results in light of haplodiploid sex determination and parasitoid mating systems, and classical biological control practices.     

Does parasitoid state affect host range expression?

The pre-release risk assessment of parasitoids for classical biological control generally involves non-target testing to define the agent’s host range. To ensure that no suitable host species are falsely rejected in these tests, it has been suggested that the physiological and informational state of parasitoids be manipulated to enhance their “motivation to oviposit”. However, the effects of such factors on host acceptance are not consistent across parasitoid species, making it laborious to identify the conditions necessary to maximise host acceptance. Our objective was to determine whether changes in parasitoid state could alter host acceptance behaviour sufficiently to affect host range expression. In addition, we tested the assumption that a state-dependent shift in motivation to oviposit on the target host will translate to a similar change in responsiveness to lower-ranked host species. Three-day-old and 10-day-old females of the candidate classical biological control agent, Diadromus pulchellus, were offered 12 non-target species of varying relatedness to the target pest, Acrolepiopsis assectella, in a series of no-choice and choice oviposition trials. Younger D. pulchellus females had previously demonstrated greater motivation to oviposit in the target pest and were, therefore, predicted to express a broader host range than older females. Parasitoid age had a minor effect on host range expression that was contrary to expectations. Older females more readily attacked one of the non-target species in no-choice tests and inflicted higher mortality in one of the choice tests. Ultimately however, young and old parasitoids still attacked the same four non-target species and their offspring emerged from the same three. There was an interaction between the effects of parasitoid condition and experimental design on responsiveness to low-ranked hosts: increasing non-target density in choice tests significantly altered attack rates by 10-day-old, but not by 3-day-old, parasitoids. The implications of these findings for host specificity testing depend largely on the specific aims of a host range assessment. Parasitoid state influenced the frequency of non-target attack but did not affect which non-target species were attacked.     

No-choice host range tests for Allotropa sp. near mecrida,a parasitoid of the pink hibiscus mealybug,Maconellicoccus hirsutus (Hemiptera: Pseudococcidae)

Host range studies of the parasitoid Allotropa sp. near mecrida (Walker) (Hymenoptera: Platygastridae) were conducted prior to applying for a permit to release it against the pink hibiscus mealybug, Maconellicoccus hirsutus (Green) (Hemiptera: Pseudococcidae), in southern California. Using M. hirsutus as a control, four mealybug species were tested as potential hosts. Allotropa sp. near mecrida did not successfully reproduce in any of the mealybug species tested, except for the target species, M. hirsutus. The parasitoid was found to host feed upon one of the non-target species within the confined test environment. Subsequently a USDA-APHIS importation and release permit was granted.     

Life history and laboratory host range of Stenopelmus rufinasus, a natural enemy for Azolla filiculoides in South Africa

The frond-feeding weevil, Stenopelmus rufinasus Gyllenhal, was imported into quarantine for testing as a potential natural enemy for the invasive fern Azolla filiculoides Lamarck in South Africa. Adult S. rufinasus lived for approximately 55 days during which the females produced on average 325 offspring. The developmental period for the immature stages (egg, three larval instars and pupation) was about 20 days indicating the potential for several overlapping generations per year. Both the adults and the larvae caused severe damage to A. filiculoides in the laboratory. Host specificity of this insect was determined by adult no-choice oviposition and larval starvation tests on 31 plant species in 19 families. Adult feeding, oviposition and larval development was only recorded on the Azolla species tested (A. filiculoides, A. pinnata subsp. poss. asiatica R.K.M. Saunders and K. Fowler, A. pinnata subsp. africana (Desv.) R.K.M. Saunders and K. Fowler and A. nilotica De Caisne Ex Mett.). A. filiculoides proved to be significantly the most suitable host for the weevil. The low adult emergence from A. nilotica and A. pinnata subsp. africana would most probably prevent the weevil from establishing on them in the field. A. pinnata subsp. poss. asiatica which supported greater development, is thought to be introduced and has a weedy phenology in South Africa and is thus of low conservation value. Therefore, any damage inflicted on this plant in the field may be an acceptable trade-off for the predicted impact of S. rufinasus on the aggressive exotic weed, A. filiculoides.     

Using parthenogenesis‐inducing Wolbachia for the selection of optimal lines of the egg parasitoid Trichogramma pretiosum for use in biocontrol

Trichogramma wasps (Hymenoptera: Trichogrammatidae) are egg parasitoids commonly employed in augmentative biological control releases against a variety of mainly lepidopteran pests. By exploiting the mechanism by which the endosymbiotic bacterium Wolbachia induces parthenogenesis in this genus, we created a set of completely homozygous Wolbachia‐infected recombinant isofemale lines (RILs), each consisting of a different combination of the genome of two well‐characterized lines of Trichogramma pretiosum Riley. We subsequently use 16 of these RILs to investigate the effect of genetic variation on various measures associated with offspring production under laboratory conditions. Unsurprisingly, substantial differences were found between the RILs in their propensity to parasitize hosts, the number of hosts they parasitize, and the levels of mortality in their offspring. Such measures can be used to choose an optimal line for biological control purposes. A method was also developed to characterize the 16 RILs using their allelic state at five loci. Essentially, this binary system uses high‐resolution melt analysis to resolve identity at each locus, with alleles originating from either the grandmaternal or grandpaternal line, and is such that each RIL can be distinguished from each other RIL by their allelic state at one or more loci. The method facilitates the easy diagnosis of line origin when two or more lines are competing with each other in competition assays, allowing for the design of more complicated tests of parasitoid quality. Future field experiments will determine which genetic line performs best under more realistic biological control conditions. The fact that these lines are infected with parthenogenesis‐inducing Wolbachia will allow for prolonged rearing without appreciable change in their genetic makeup, which should lead to a predictable biological control performance.     

Selecting the right parasitoid for the environment in classical biological control programmes: the case of Diadegma semiclausum (Hymenoptera: Ichneumonidae) and Plutella xylostella (Lepidoptera: Plutellidae) in the Kofele highland of Ethiopia

The careful selection of parasitoids with a track record of success in similar environmental conditions may increase the likelihood of effective biocontrol. Diamondback moth (DBM) population and level of parasitism were assessed for three years prior to the release of Diadegma semiclausum (Hellén) in June 2008. Thereafter, the DBM population and severity of crop damage and the occurrence of D. semiclausum and indigenous DBM parasitoids were monitored by monthly surveys during the main production season of June to December for three years. Before release, DBM numbers fluctuated between 4.2 and 11.2 per plant and parasitism ranged between 6.5 and 24.7%. DBM density declined to 2.8, 0.9 and 0.7 per plant whilst parasitism levels increased successively to 21, 39 and 38% in 2008, 2009 and 2010, respectively. A significant shift in parasitoid guild was observed after D. semiclausum release: it comprised 18.6, 80.3 and 88.6% of total parasitism in the first, second and third year, respectively. The decline of DBM density following the release of the introduced parasitoid and its establishment will ensure production of Brassica spp. without pesticide use against DBM in the affected area.     

Host range testing of the nearctic beneficial parasitoid <Emphasis Type="Italic">Neodryinus typhlocybae</Emphasis>

Neodryinus typhlocybae (Ashmead) (Hymenoptera: Dryinidae) is a natural enemy of the planthopper Metcalfa pruinosa (Say) (Hemiptera: Flatidae), introduced from North America into Europe and regionally established as a pest species. Prior to possible utilization of the parasitoid as a biocontrol agent in Austria, its potential negative impacts on eight native plant- and leaf-hopper species were examined in the laboratory. Non-target species were selected according to the following criteria (a) occurrence in Austria, (b) close phylogenetic relationship with M. pruinosa, (c) larvae free-living and surface-dwelling, (d) phenology, (e) larval size, (f) ecological similarity with M. pruinosa and (g) availability of sufficient numbers of individuals. The Auchenorrhyncha species Issus coleoptratus (Fabricius), Chloriona smaragdula (St?l), Conomelus anceps (Germar), Alebra wahlbergi (Boheman), Empoasca sp., Idiocerus stigmaticalis (Lewis), Macrosteles septemnotatus (Fallén) and Japananus hyalinus (Osborn) were chosen for testing. Larvae from both the target and the non-target species were offered separately to N. typhlocybae females in no-choice laboratory tests and all attacks, instances of host feeding and parasitizations were recorded. No non-target species was attacked, fed upon or parasitized by N. typhlocybae, whereas M. pruinosa was attacked frequently. This study supports the assumption that the host range of N. typhlocybae is restricted to Flatidae, of which only the introduced species occurs in Austria. Direct negative effects on other Auchenorryncha species in Austria are therefore unlikely to occur.

Adult feeding and lifetime reproductive success in the parasitoid Aphytis melinus

The diet of adult females of the parasitoid Aphytis melinus DeBach (Hymenoptera: Aphelinidae) includes host insects and sugar-rich foods such as nectar and honeydew. We compared the contributions of host feeding to longevity and fecundity in A. melinus females in the presence and in the absence of honey meals. First, we assessed the longevity of females that were not allowed to oviposit. While the longevity of females fed honey was significantly increased by host feeding (median ages were 30.5 days for host-fed females and 17 days for females not allowed to host feed), the lifespan of parasitoids not fed honey did not exceed 3 days for any individual and there was no effect of host feeding on longevity in this group. In the second set of experiments, we assessed the fecundity and longevity of females allowed to oviposit. We conducted two experiments, one in which honey was continuously available, and one in which honey was not available. In both experiments, daily observations were made of females that were either allowed to host feed or manually prevented from host feeding. In the presence of honey, host feeding significantly increased both fecundity and longevity, and in the absence of honey, parasitoids died within 2 days and host feeding had no significant effect on either fecundity or longevity. The lifetime fecundity of females fed honey but not hosts exceeded the initial egg complement by 60% on average. Approximately one host per day was used for host feeding whether honey was supplied or not, and each host-feeding meal contributed approximately 3.9 eggs to the lifetime fecundity of honey-fed females. In the last experiment, we compared the rate of egg resorption over a 36-h period in A. melinus females that were deprived of hosts and either fed honey or starved. While no egg resorption was detected in honey-fed females over this time period, starved females resorbed approximately 9 eggs. Thus, the availability of a sugar-rich food interacts strongly with host feeding in influencing longevity and fecundity and has a strong direct effect on egg resorption.     

Life cycle and host range of Phycita sp. rejected for biological control of prickly acacia in Australia

Prickly acacia (Vachellia nilotica subsp. indica), a native of the Indian subcontinent, is a serious weed of the grazing areas of northern Australia and is a target for classical biological control. Native range surveys in India identified a leaf webber, Phycita sp. (Lepidoptera: Pyralidae) as a prospective biological control agent for prickly acacia. In this study, we report the life cycle and host‐specificity test results Phycita sp. and highlight the contradictory results between the no‐choice tests in India and Australia and the field host range in India. In no‐choice tests in India and Australia, Phycita sp. completed development on two of 11 and 16 of 27 non‐target test plant species, respectively. Although Phycita sp. fed and completed development on two non‐target test plant species (Vachellia planifrons and V. leucophloea) in no‐choice tests in India, there was no evidence of the insect on the two non‐target test plant species in the field. Our contention is that oviposition behaviour could be the key mechanism in host selection of Phycita sp., resulting in its incidence only on prickly acacia in India. This is supported by paired oviposition choice tests involving three test plant species (Acacia baileyana, A. mearnsii and A. deanei) in quarantine in Australia, where eggs were laid only on prickly acacia. However, in paired oviposition choice trials, only few eggs were laid, making the results unreliable. Although oviposition choice tests suggest that prickly acacia is the most preferred and natural host, difficulties in conducting choice oviposition tests with fully grown trees under quarantine conditions in Australia and the logistic difficulties of conducting open‐field tests with fully grown native Australian plants in India have led to rejection of Phycita sp. as a potential biological control agent for prickly acacia in Australia.     

Harmonization of regulations for invertebrate biocontrol agents in Europe: progress,problems and solutions

The use of non‐native invertebrate biological control agents (IBCAs) in Europe is not covered by a Directive equivalent to that which regulates biocontrol with microorganisms or the genetic modification of crop plants. Regulation is at the discretion of individual member states and largely derived from national legislation on pesticides, plant health or environmental protection. There is no EU country with regulation of IBCAs that requires information on the microbial symbiont content of candidate species, and in the absence of horizontal transfer under natural conditions, this policy is unlikely to change. Although there have been few reported negative effects linked to the import and release of IBCAs, a number of countries have introduced or revised their regulatory frameworks in recent years. This article reviews major developments in the regulation and environmental risk assessment (ERA) of IBCAs in Europe over the last 10 years including: the fragmented pattern of regulation between countries, variation in information requirements for release licences, format and methods of ERA for different taxonomic groups of IBCAs, use and updating of the European Plant Protection Organisation Positive List, sources of expert advice on ERA data, communication between IBCA regulators, and options for the provision of international leadership to coordinate regulatory and ERA‐related issues with IBCA‐based biocontrol in Europe.     

Biology and host range of Ophiomyia camarae Spencer (Diptera: Agromyzidae), a potential biocontrol agent for Lantana spp. (Verbenaceae) in Australia

The life history and host range of the herringbone leaf-mining fly Ophiomyia camarae, a potential biological control agent for Lantana spp., were investigated. Eggs were deposited singly on the underside of leaves. Although several eggs can be laid on a single leaf and a maximum of three individual mines were seen on a single leaf, only one pupa per leaf ever developed. The generation time (egg to adult) was about 38 days. Females (mean 14 days) lived longer than males (mean 9 days) and produced about 61 mines. Oviposition and larval development occurred on all five lantana phenotypes tested. Eleven plant species representing six families were tested to determine the host range. Oviposition and larval development occurred on only lantana and another nonnative plant Lippia alba (Verbenaceae), with both species supporting populations over several generations. A CLIMEX model showed that most of the coastal areas of eastern Australia south to 30°16′ S (Coffs Harbour) would be suitable for O. camarae. O. camarae was approved for release in Australia in October 2007 and mines have been observed on plants at numerous field sites along the coast following releases.     

Unsuitability of Charidotis pygmaea for biocontrol of Lantana camara in Africa

The host range of the tortoise beetle,Charidotis pygmaea Klug (Coleoptera:Chrysomelidae), was studied under quarantinelaboratory conditions to evaluate the insect'ssuitability for release as a biological controlagent for the noxious weed, Lantanacamara L. (Verbenaceae) in South Africa.Culturing on the target plant, L. camara,proved problematic with high larvalmortalities. Host-specificity studies showedthat four species in the genus Lantana,and two species in the genus Lippiawere acceptable as host plants. Duringlarval development trials, the insect performedbetter on the indigenous Lantana rugosaThunb. (Verbenaceae) and the introduced,commercially used L. montevidensis(Spreng.) Briq. (Verbenaceae), than on any ofthe weedy South African L. camaravarieties tested. Adult multi-choice trialsindicated that the beetle preferred to ovipositon L. rugosa and L. montevidensis.It is therefore recommended that C.pygmaea not be released against L.camara in Africa.     

Biology and host range of the stem-boring beetle Aphanasium australe, a promising agent for the biological control of Hakea sericea in South Africa

The invasive Australian shrubHakea sericea Shrader is a majorenvironmental weed in the Western and EasternCape Provinces of South Africa. Dense,impenetrable thickets severely threaten theunique endemic vegetation of the Cape FloristicKingdom, increase fire hazards and reduce wateryields in catchments. Biological control,initiated in the 1970s, is largely confined tothe use of seed-feeding insect agents. Becausenone of these agents reduce the density ofexisting hakea populations, a stem-boringbeetle, Aphanasium australe (Boisduval) (Cerambycidae),was imported into quarantine in South Africa in1975. During multichoice oviposition tests,involving 12 Australian and six South Africanproteaceous species, in a walk-in cage, A. australe only oviposited on four species ofHakea and two exotic species of Grevillea. However, culturing difficultiesresulted in the suspension of host-specificitytesting after three years. Testing was resumedfollowing re-importations in 1995, also becauseA. australe also attacks Hakeagibbosa (Sm.) Cav., which is not attacked byany of the existing biocontrol agents. Duringno-choice survival tests, involving 66test plant species from 15 families, A. australe only developed on H. sericea, H. gibbosa and one exoticspecies of Grevillea. The contention thatA. australe is confined to the genus Hakea was confirmed by host records andsurveys in Australia which provided no evidenceof attacks on crop, pasture or related plants.The regulatory authorities accepted theseresults and A. australe was cleared forrelease in South Africa during 2001.     

Biology and host preference of the planthopper Taosa longula (Hemiptera: Dictyopharidae), a candidate for biocontrol of water hyacinth

Taosa longula Remes Lenicov (Hemiptera: Dictyopharidae) is a planthopper from the South American tropics that feeds on water hyacinth, Eichhornia crassipes (Mart.) Solms-Laubach (Pontederiaceae). The biology of T. longula was studied in the laboratory and field to evaluate it as a potential biological control agent for this widespread aquatic weed. The developmental time of nymphs was recorded at different temperatures (15, 19, 23, 25, 27 and 30 °C), and developmental threshold temperatures were obtained for the different instars. The host range was evaluated in terms of development and feeding preference. Development from instar I to adult was recorded in two no-choice trials, one with cut leaves of Pontederiaceae, and a second with growing whole plants. In the cut-leaf tests, adults were obtained from Pontederia cordata var. cordata, P. rotundifolia and water hyacinth. In the whole plant test, T. longula adults were obtained only from water hyacinth. Feeding preference was evaluated by means of a paired-choice test with 10 T. longula first instars on whole plants of P. c. cordata, P. rotundifolia and water hyacinth. The number of insects that fed on water hyacinth was significantly higher than on P. c. cordata and P. rotundifolia. Taosa longula showed a clear preference for water hyacinth and exhibited warm climate requirements, making it an attractive candidate for water hyacinth biological control in tropical and subtropical areas.     

Host range and risk assessment of Zygogramma bicolorata,a defoliating agent released in South Africa for the biological control of Parthenium hysterophorus

Parthenium hysterophorus (parthenium) is a weed of international importance and is spreading rapidly in sub-Saharan Africa. Consequently, it has been targeted for biocontrol in South Africa since 2003. Based on precedents elsewhere in the world, the defoliating beetle Zygogramma bicolorata was prioritised as a candidate agent. Although no-choice tests, involving some 48 test plant species, indicated a significant preference for parthenium, significantly reduced feeding and oviposition was recorded on some species. Multiple-choice tests resolved many of these non-target results; however, Helianthus annuus (sunflower) was still selected for oviposition and feeding. Of the 12 sunflower cultivars tested, four were selected for oviposition, while two were selected for oviposition and feeding. These six cultivars were then subjected to larval development trials, together with three native and two weed species (in the Asteraceae). These trials showed high levels of complete development on parthenium, significantly reduced development on sunflower cultivars, and partial development on only one of the weed species. Finally, a risk assessment was conducted on the six sunflower cultivars to quantify Z. bicolorata feeding and reproductive performance. Feeding risk calculations revealed these cultivars to have an extremely low risk (<0.2%) of supporting Z. bicolorata feeding and development. Similarly, reproductive risk calculations showed a very low risk (<0.16%) of supporting viable Z. bicolorata populations. These data are supported by findings from both the native (Mexico) and introduced ranges (Australia, India) of Z. bicolorata, where it has never been recorded as a pest of sunflower. These considerations were accepted by the regulatory authorities and in August 2013, Z. bicolorata became the second insect agent to be released in South Africa for the biocontrol of parthenium.     

The host range and biology of <Emphasis Type="Italic">Cometaster pyrula</Emphasis>; a biocontrol agent for <Emphasis Type="Italic">Acacia nilotica</Emphasis> subsp. <Emphasis Type="Italic">indica</Emphasis> in Australia

Prickly acacia, Acacia nilotica subsp. indica (Benth.) Brenan, a major weed of the Mitchell Grass Downs of northern Queensland, Australia, has been the target of biological control projects since the 1980s. The leaf-feeding caterpillar Cometaster pyrula (Hopffer) was collected from Acacia nilotica subsp. kraussiana (Benth.) Brenan during surveys in South Africa to find suitable biological control agents, recognised as a potential agent, and shipped into a quarantine facility in Australia. Cometaster pyrula has a life cycle of approximately 2 months during which time the larvae feed voraciously and reach 6 cm in length. Female moths oviposit a mean of 339 eggs. When presented with cut foliage of 77 plant species, unfed neonates survived for 7 days on only Acacia nilotica subsp. indica and Acacia nilotica subsp. kraussiana. When unfed neonates were placed on potted plants of 14 plant species, all larvae except those on Acacia nilotica subsp. indica and Acacia nilotica subsp. kraussiana died within 10 days of placement. Cometaster pyrula was considered to be highly host specific and safe to release in Australia. Permission to release C. pyrula in Australia was obtained and the insect was first released in north Queensland in October 2004. The ecoclimatic model CLIMEX indicated that coastal Queensland was climatically suitable for this insect but that inland areas were only marginally suitable.     

Megamelus scutellaris (Hemiptera: Delphacidae), a biocontrol agent of water hyacinth,is not sufficiently specific for release in Australia

Water hyacinth Eichhornia crassipes (Pontederiaceae) is one of the world's worst invasive species, responsible for damaging aquatic systems in many warmer parts of the globe including north America, Africa, Asia and Australia. The planthopper Megamelus scutellaris Berg (Delphacidae) has been released in USA and approved for release in South Africa for biocontrol of water hyacinth. We assessed this agent for suitability for release in Australia and found that a related native aquatic plant, Monochoria cyanea (Pontederiaceae) is within the fundamental host range of this insect. Adult survival, oviposition and development of nymphs to adult was equally high on M. cyanea as on the target species, although the quality of these next generation adults was lower than those reared on the target species. This demonstrates that M. scutellaris is not sufficiently specific for release in Australia. Nymphal development to adults occurred only in very low numbers on the three other Australian species of Monochoria. M. cyanea only occurs in Australia so M. scutellaris is still a possible water hyacinth biocontrol candidate for other regions depending on the results of assessment of the risk to local species of Monochoria. This study demonstrates the effectiveness of modern biocontrol agent assessment and reinforces the importance of testing of local non-target species.     

Pre‐release risk assessment of the egg‐parasitoid Gryon pennsylvanicum for classical biological control of Leptoglossus occidentalis

Leptoglossus occidentalis Heidemann (Heteroptera: Coreidae) is a North American conifer seed pest that was accidentally introduced to Europe. In the Mediterranean area, it threatens the production of Pinus pinea Linnaeus seeds. The egg‐parasitoid Gryon pennsylvanicum (Ashmead) (Hymenoptera: Platygastridae), the main natural enemy in the native range of L. occidentalis, was imported from British Columbia to Italy. Pre‐release risk assessments were made under quarantine conditions by no‐choice tests conducted with naïve and experienced G. pennsylvanicum offering single eggs of target and non‐target species for varying exposure times (1, 4, 48 h). G. pennsylvanicum successfully parasitized from 75% to 100% of the target host eggs. Only one female specimen of the egg‐parasitoid emerged from a non‐target egg (Gonocerus juniperi Herrich‐Schaeffer, Heteroptera: Coreidae). Two dead female specimens were found, one inside an egg of Coreus marginatus (Linnaeus) (Heteroptera: Coreidae) and one in an egg of Camptopus lateralis (Germar) (Heteroptera: Alydidae). All three cases occurred at the longest oviposition exposure time. Results obtained with this conservative approach suggest that the risk to non‐target species of releasing G. pennsylvanicum in Italy is low.     

Safety assessment of biocontrol and plant growth‐promoting pseudomonads useful in crop production

One biocontrol and two plant growth‐promoting Pseudomonas spp. isolates were subjected to a safety assessment. Potential risks for human and plant health were investigated and screenings for toxic effects were performed. The antibiotic susceptibility pattern was typical for Pseudomonas and only one of the isolates grew at 37°C. None of the isolates elicited a hypersensitivity reaction in the tobacco test for plant pathogenicity. For toxicity testing, BACTOX, the Lemna growth bioassay, primary root and shoot growth in vitro and a seed germination/early seedling growth assay were performed. In these assays, one of the plant growth‐promoting isolates consistently displayed concentration‐dependent adverse effects not seen with the other isolates. Further investigation is needed to determine whether these adverse effects are a concern from a safety assessment perspective, as the identity and mode of action of the active metabolite(s) are unknown. Lack of standardised test procedures for complex samples of microbial origin hampered interpretation of the results from the toxicity assays and there is a need to develop methodology that is more suitable for testing such samples. Nevertheless, the tests employed for the three Pseudomonas isolates were successful in distinguishing isolates with different characteristics. This test framework provides an outline for information collection and safety evaluation when handling new microbial isolates that could be an efficient tool in selecting the best candidate isolates for product development.