BioControl (2019) 64:457–468 https://doi.org/10.1007/s10526-019-09946-0 (0123456789().,-volV)( 0123456789().,-volV) Post-release evaluation of a combination of biocontrol agents on Crofton weed: testing extrapolation of greenhouse results to field conditions Lisa Buccellato . Marcus J. Byrne . Jolene T. Fisher . Ed T. F. Witkowski Received: 17 January 2019 / Accepted: 24 June 2019 / Published online: 3 July 2019 Ó International Organization for Biological Control (IOBC) 2019 Abstract Two biocontrol agents, a leaf-spot patho- growth). The greenhouse trails were therefore not gen, Passalora ageratinae, and a stem gall fly, predictive of field conditions. Procecidochares utilis, have been released against Crofton weed, Ageratina adenophora (syn. Eupato- Keywords Ageratina adenophora Á Crofton weed Á rium adenophorum) (Asteraceae), in South Africa. Insect-plant-pathogen interaction Á Multiple This work reports the first post-release evaluation of biocontrol agents Á Passalora ageratinae Á the effect of both agents acting together in the field. A Procecidochares utilis greenhouse trial using both agents had predicted an additive (beneficial) interaction between the agents. This study investigated if the additive interaction was Introduction present in the field. Four month old stems were exposed to one of the following three treatments Classical biocontrol of invasive alien plants involves (n = 20 plants per treatment): pathogen-only, patho- the deliberate introduction of agents, such as insects gen plus single fly-galled, and pathogen plus double and pathogens, to negatively influence the invasive fly-galled, for 11 months. The interaction between the plant’s growth parameters and population dynamics agents was equivalent to both agents acting indepen- (Mu¨ller-Scha¨rer and Schaffner 2008; Morin et al. dently (i.e. there was no additive effect on the weed’s 2006; Zachariades et al. 2017). Often, more than one biocontrol agent is released onto a specific target weed, based on the theory that the combined effects of multiple biocontrol agents will increase control of the Handling Editor: S. Raghu weed (Denoth et al. 2002; Julien and Griffiths 1998; L. Buccellato Á M. J. Byrne Á J. T. Fisher (&) Á Myers 1985, 2008; Stephens et al. 2013; Stiling and E. T. F. Witkowski Cornelissen 2005). Synergistic relationships between School of Animal Plant and Environmental Sciences, insect and pathogen agents are considered responsible University of the Witwatersrand, PO Wits, Johannesburg 2050, South Africa for some of the first successes in biocontrol, of e-mail: [email protected] Opuntia stricta and of Hypericum perforatum for example (Caesar 2000, 2003). M. J. Byrne However, there is an increasing call not to release DST-NRF Centre of Excellence for Invasion Biology, School of Animal, Plant and Environmental Sciences, agents which are unlikely to be very effective (Paynter University of the Witwatersrand, et al. 2018; Raghu et al. 2006) because of the risks PO Wits, Johannesburg 2050, South Africa 123 458 L. Buccellato et al. associated with non-target effects. Therefore in addi- natural vegetation (Erasmus et al. 1992; Henderson tion to host specificity, the effectiveness of biocontrol 2001; Niu et al. 2007; Poudel et al. 2019). Crofton agents is often assessed under controlled conditions in weed also reduces crop yield, the carrying capacity of laboratory or greenhouse studies (Cowie et al. 2017; grazing land, is unpalatable to cattle, and causes a fatal Kumaran et al. 2018; Morin et al. 2009). However, lung disease in horses (Land Protection 2001; Page under such controlled conditions the impact of the and Lacey 2006; Plant Protection News 1988). agents can be either under- or over-estimated, as these In 1984 a biocontrol programme for the control of are not always representative of conditions in the field Crofton weed was initiated in South Africa (Plant (Morin et al. 2006, 2009; Rosskopf et al. 1999). For Protection News 1987; Kluge 1991). Two biocontrol example, the Eucalyptus weevil, Gonipterus ‘‘scutel- agents, a stem gall fly, Procecidochares utilis Stone latus’’ Gyllenhal (Curculionidae), was more selective (Tephritidae), and a leaf-spot pathogen, Passalora in its feeding and oviposition behaviour on Eucalyptus ageratinae Crous and A.R. Wood (Mycosphaerel- species in the field in comparison to laboratory trials laceae) (previously named Cercospora eupatorii Peck (Newete et al. 2011) highlighting potential differences or Phaeoramularia sp.) (Crous et al. 2009), were in the predicted versus realised host ranges of released in South Africa in 1984 and 1987, respec- biocontrol agents. Biocontrol risk assessment gener- tively (Kluge 1991). The biology and impact of the fly ally centres on the dangers of potential non-target in Australia have been described by Haseler (1965) feeding of the putative agent (Blossey et al. 2018). and in South Africa by Bennett (1986). The pathogen’s However, post-release evaluations of released agents host specificity was explored by Morris (1989) and are critical for the future of biocontrol (Morin et al. Wang et al. (1997) who measured its impact in 2009; Raghu et al. 2006), not least to continuously Australia. Heystek et al. (2011) reviewed the Crofton reassure the public that it is a safe and cost-effective weed biocontrol programme in South Africa and pest control method (Messing and Brodeur 2018), but concluded that additional agents should be sought. also to inform politicians that government investment However, this is the first post-release evaluation of in the technique is money well spent (Ivey et al. 2019; both agents’ impact when released together in the field Schwarzla¨nder et al. 2018a). In light of this, this study in South Africa. assesses the impact of two biocontrol agents in the A controlled greenhouse trial using both the fungal field, released against Ageratina adenophora, follow- pathogen and the gall fly, individually and in combi- ing on from greenhouse trials of efficacy of the two nation, showed that the fly reduced plant height and biocontrol agents acting together. growth of Crofton weed, and repeated galling by the Originating from Mexico, Ageratina adenophora fly was most detrimental to the weed (Buccellato et al. (Sprengel) King and Robinson (syn. Eupatorium 2012). The pathogen did, however, reduce the number adenophorum Spreng.) (Asteraceae), also known as of sideshoots, supressing vegetative reproduction of Crofton weed or the Mexican Devil, is an invasive the weed, which was unfortunately promoted by weed in several countries worldwide, including South galling (Buccellato et al. 2012). Nevertheless, the Africa, Australia, New Zealand, Hawaii, India, China, combination of the two agents resulted in an additive and recently in Italy (Julien and Griffiths 1998; Poudel effect on control of Crofton weed in the greenhouse. et al. 2019). Crofton weed is a perennial herb, with a The categorisation of combined impacts of the agents woody rootstock, and many stems reaching up to 2 m follows those proposed by Hatcher and Paul (2001), in height (Bess and Haramoto 1959; Henderson 2001; and Turner et al. (2010). Page and Lacey 2006). Trailing Crofton weed stems In South Africa, anecdotal field observations sug- produce roots when in contact with the soil, resulting gested that neither the fly nor fungal pathogen are in dense infestations (Bess and Haramoto 1959; successfully controlling Crofton weed (Heystek et al. Morris 1991). The plant invades steep slopes and 2011). However, the impact of the two agents together wet areas along streams, roadsides, forests, and has not been formally assessed in the field in South plantations (Dodd 1961; Henderson 2001; Page and Africa. The aim was therefore to evaluate whether the Lacey 2006; Trounce 2003). Conservation areas are pathogen and fly in combination will control the adversely affected by this weed, as it is allelopathic, vegetative growth of Crofton weed under field alters soil microbial communities, and displaces 123 Post-release evaluation of a combination of biocontrol agents on Crofton weed 459 conditions, in South Africa, as predicted by the In June 2007, 20 plants were randomly allocated to greenhouse studies. each of the following three treatments: (1) plants infected with the fungal pathogen, P. ageratinae (pathogen-only); Materials and methods (2) plants infected with the fungal pathogen, P. ageratinae, and exposed to one release of the Field site fly, P. utilis (hereafter referred to as pathogen- single galled); A field trial was conducted on the 150 ha Kloofwaters (3) plants infected with the fungal pathogen, P. farm (1380 m a.s.l.), in the Magaliesberg, North West ageratinae, and exposed to two releases of the Province, South Africa (25°49045.100S, 27°26026.000E). fly, P. utilis (hereafter referred to as pathogen- The farm is privately owned, and predominantly used double galled. Pathogen-double galled 1st gen- for school camps. However, a low stocking level of eration refers to the first fly release, and 2nd cattle is present. The field site was located within generation refers second fly release). Crofton weed infestations along a flat stream embank- ment, facing eastwards. Plants were in full sunshine, Cages were used to restrict flies from ovipositing on with late afternoon shading and only the fungal non-galled treatment plants. The cages were con- pathogen, P. ageratinae, was present. The site is on structed from fine white netting, attached to a 1 m Moot Plains Bushveld (central bushveld, Savanna central pole with a steel ring at the top, and pegged into Biome), with thorny savanna, dominated by Acacia the soil. All plants in all treatments were covered with species and woodlands (Mucina et al. 2006; Ruther- cages. Cages were removed two weeks after flies were ford et al. 2006). The Magaliesberg region experiences released, as the average life span of the adult flies is summer rainfall and very dry winters with frost ten days. No galling on pathogen-only stems was (Mucina et al. 2006). In winter, temperatures ranged observed during the trial period. from 2.6 to 18.3 °C (mean = 9.9 °C), and from 14.0 to Adult P.