Conventional willow control in wetland and riparian areas of New Zealand is undertaken using cut-stump and/or drill and injection application of glyphosate. The presence of herbicide residues in catchment water supplies has seen the investigation of non-chemical alternatives to poplar and willow control in Auckland water catchment areas. We have demonstrated, in glasshouse trials, the efficacy of an aqueous, gel-based formulation of Chondrostereum purpureum to control the regrowth of crack and grey willow (Salix fragilis and S. cinerea). Chondrostereum purpureum isolate ICMP 16392 (isolated from a Prunus sp.) produced the fastest biomass accumulation in liquid culture. Crack willow was significantly more susceptible to cut-stump infection by C. purpureum than grey willow in the glasshouse trial at the end of the 23-week period. Two different formulations were tested; at the end of the trial, there was no significant difference between them with respect to monthly biomass accumulation. Successful field applications of C. purpureum through cut and paste and drill and injection were confirmed by the presence of fruiting bodies on both treated species. Resprouting ability as measured by shoot number was significantly lower on C. purpureum inoculated stumps.

Eleven plant species representing two native ecosystems (1) podocarp forest (Dacrycarpus dacrydioides, Plagianthus regius, Pittosporum eugenioides, Cordyline australis, Melicytus ramiflorus, Coprosma robusta and Asplenium gracillimum) and (2) grassland (Poa cita, Chionochloa rubra, Chionochloa rigida and Festuca novae-zelandiae) and representatives of a wider beneficial microbe population (arbuscular mycorrhizae (AM) and Pseudomonas spp.) were studied to assess possible non-target effects of a commercial T. atroviride product. Comparison of several physical markers (plant height, basal diameter, total leaf number, total leaf area, and fresh and dry weight of leaves, shoots and roots) showed that this T. atroviride isolate had no negative effect on plant health. Although photosynthetic pigment analysis indicated significant differences in chlorophyll and carotene levels between the Trichoderma and control treatments for some plants, this variation was not supported by physical changes in plant health. Culture-dependent and -independent analysis of AM fungi and Pseudomonads demonstrated that T. atroviride had no effect on these potential plant beneficial taxa in either ecosystem. The findings from this study suggest using Pittosporum eugenioides and Pagianthus regius for assessing the impact of imported microbial biocontrol agents on the plant growth of established podocarp forest plants, and Cordyline australis and P. regius for assessing the impact of these agents on seedling establishment. In tussock grassland ecosystems, Festuca novae-zelandiae and Poa cita are suggested for growth impact assessments, and P. cita and Chionochloa rubra for seedling establishment trials. The use of a combination of basic culture-dependent and -independent techniques for assessing changes in soil microbial communities often associated with plant health is also suggested.

The introduction of Gaeumannomyces graminis var. tritici (Ggt) inoculum into soils to screen for take-all suppression has been widely used in field and laboratory studies. However, the amounts of Ggt inoculum reported have varied greatly. The effects of adding Ggt in sand/maizemeal to three soils of different cropping history at the rates of 0, 0.2, 0.5, 1 and 4 % (w/w) were investigated in a pot assay using wheat plants. The three soils had previously been cropped with ryegrass for 5 years, wheat for 8 years, and wheat for 2 years. The soils represented a putative non-suppressive, non-wheat soil; a suppressive wheat soil; and a non-suppressive wheat soil, each containing natural background concentrations of Ggt DNA of 0, 200 and 1126 pg g−1 soil, respectively. Root assessments of wheat plants after 4 weeks growth showed that 4 % of Ggt reduced root growth slightly, decreased water uptake of the wheat plants and effectively differentiated the suppressive activity of the soils (P < 0.01, take-all incidences of 83, 69 and 81 %, respectively), and was therefore suitable for investigating take-all suppression in soils.

Lynley Hayes1, Simon V. Fowler1, Quentin Paynter2, Ronny Groenteman1, Paul Peterson3, Sarah Dodd2, Stanley Bellgard2 1 Landcare Research, PO Box 69040, Lincoln 7640, New Zealand 2 Landcare Research, Auckland, New Zealand 3 Landcare Research, Palmerston North, New Zealand ABSTRACT: New Zealand has a serious problem with unwanted exotic weeds. Invasive plants threaten all ecosystems and have undesirable impacts on primary production and biodiversity values, costing the country billions of dollars each year. Biocontrol is a key tool for reducing the impacts of serious, widespread exotic weeds. We review the nearly 90-year history of weed biocontrol research in New Zealand. Thirty-eight species of agents have been established against 17 targets. Establishment success rates are high, the safety record remains excellent, and support for biocontrol remains strong. Despite the long-term nature of this approach partial control of fi ve targets (Mexican devil weed Ageratina adenophora, alligator weed Alternanthera philoxeroides, heather Calluna vulgaris, nodding thistle Carduus nutans, broom Cytisus scoparius), and good control of three targets (mist fl ower Ageratina riparia, St John’s wort Hypericum perforatum, and ragwort Jacobaea vulgaris) have already been achieved. The self-introduced rust Puccinia myrsiphylli is also providing excellent control of bridal creeper Asparagus asparagoides. Information about the value of successful weed biocontrol programmes is starting to become available. Savings from the St John’s wort project alone have more than paid for the total investment in weed biocontrol in New Zealand to date. Recent research advances are helping us to select the best weed targets and control agents, and are enabling biocontrol programmes to be even safer and more effective. Future challenges include expanding the range of targets to include more aquatic species, fi nding ways to do more for less given the number of weeds needing to be controlled, and developing bioherbicides through to commercially available products. The implications of climate change need to be kept in mind, but fortunately seem unlikely to substantially disrupt biocontrol programmes because biocontrol agents should be able to follow changes in weed distributions.

In the native range of Tradescantia fluminensis in SE Brazil surveys revealed a natural enemy biota attacking the plant that was rich in potential biocontrol agents for New Zealand (NZ), including nine fungi and 10 herbivorous insect species. Similar surveys in NZ, where T. fluminensis is an invasive exotic weed, revealed no specialist insect herbivores or pathogens. The Brazilian insect herbivores included leafmining, stemboring and gall-forming feeding guilds that were absent in NZ. Mean foliar damage levels per site on T. fluminensis were 7.8× higher for folivores in Brazil cf. NZ, and 21.2× higher for pathogens. The presence of rust pustules, or 'brown lesions', on leaves in Brazil was negatively associated with damage by folivores, perhaps indicating an antagonistic interaction. In contrast, damage by the white smut fungus, Kordyana sp., was not negatively associated with folivore damage. Mean dry biomass of T. fluminensis was significantly lower in Brazil (164 g m−2) cf. NZ (455 g m−2). In NZ, 85% of sites had biomass measures >200 g m−2 (the previously determined threshold above which native forest regeneration fails). In Brazil, only 27% of sites had biomass measures >200 g m−2. Among the insect herbivores, three chrysomelid beetles, Neolema ogloblini, Neolema abbreviata and Lema basicostata were prioritised as potential biocontrol agents. Their larvae cause potentially complementary damage to leaves, shoot-tips and mature stems, respectively. Several pathogens, including a rust, were rejected before we selected the Kordyana species. Host range testing of all four agents showed sufficient host-specificity for consideration for release in NZ. Neolema ogloblini and L. basicostata were field-released in NZ in 2011 and 2012, with the field-release of N. abbreviata due late 2012. An application to release Kordyana sp. in NZ has been made.

Many ecologists profess a negative opinion of biocontrol, whilst practitioners argue that it offers a cost-effective solution for many invasive weed problems. Practitioners are under pressure to implement effective weed biocontrol more quickly, cheaply and safely. In our practitioner's perspective, we focus on two key areas, host range testing and indirect non-target effects, where advances in ecological research could progress these stakeholder-driven aims and minimize potential negative outcomes of biocontrol that concern ecologists and practitioners. Host specificity testing is used to discard weed biological control agents that might damage native or valued exotic plants. 'No-choice' starvation testing is almost infallible at identifying plants that cannot be hosts and those which have the potential to support development of a biocontrol agent – which defines the fundamental host range. However, such tests can produce 'false positives' and reject agents that, under real host selection situations in the field, cause no direct non-target effects. For example, Chrysolina spp. beetles are the basis for New Zealand's most successful weed biocontrol programme but would probably not be released nowadays because retrospective laboratory host range testing demonstrates that indigenous Hypericum spp are within the agents' fundamental host ranges. Nevertheless, recent field studies have not shown significant impacts on these indigenous congenerics (Groenteman, Fowler & Sullivan 2011). This example is far from unique: many successful weed biocontrol programmes in the past relied on agent species that would not pass modern regulatory scrutiny despite there being no evidence of harmful post-release non-target effects (H. Hinz, personal communication). For the future then, unless we improve our risk assessment, we will reject potentially successful and safe agents based on overly conservative host range testing. In reality, determining the realised host range (i.e. the plant species that will support agent populations in the field) has proven difficult. Previously, if laboratory tests showed that an agent could develop from egg to adult on a test plant, it was believed that choice tests would better predict the risk of attack on that plant species in the field. This approach has proven unreliable. The ideal testing option is open-field specificity tests conducted in the native range, but these cannot always be performed (e.g. quarantine restrictions may prohibit importing key test plants into the native range of a weed). We need a framework for assessing whether a fundamental host is likely to become a significant realised host without having to rely on native-range open-field specificity testing. Choice tests have failed to assess the realised host range of agents such as seed feeders, where asynchrony between an agent's activity and host plant availability can result in 'no-choice' situations in the field (e.g. Paynter et al. 2008). Are choice tests reliable for agents that attack non-ephemeral host plant structures (e.g. stems or roots) where phenological asynchrony is unlikely, or can non-target attack result from 'no-choice' situations arising when agents disperse away from their normal hosts? Could investigating the influence of deprivation on no-choice testing results predict whether a non-target host is likely to be permanently colonised or only subject to spillover attack (Withers 1997)? More, well-studied examples are needed. In some cases, it may be relatively simple to predict when non-target attack is likely to be minor spillover: Taylor et al. (2007) found that the native leguminous shrub Neptunia major (Benth.) Windler supported the development of the continuously brooded biocontrol agent Neurostrota gunniella (Busck) that targeted the invasive weed Mimosa pigra L. However, N. major died back during the dry season. Therefore, N. gunniella populations could not persist on this native host, and only those individuals growing close to M. pigra were regularly attacked. Evidence of past host shifts can be seen in the phylogenies of close relatives of biocontrol agents. With some rusts, past host range expansions have even led to the fungus exploiting taxonomically unrelated alternative hosts, often as part of complex life cycles. Do past evolutionary events indicate a higher risk of host range expansions/shifts? Could host range testing help to assess this risk by revealing a 'ghost of past evolutionary adaptation' to other, sometimes taxonomically distant, host plant taxa? The host specificity of biocontrol agents can vary across their native ranges. Does this suggest a greater risk of post-release host range expansion even if only monophagous populations were used to source agents? Would modern host range testing show that the fundamental host range reflected the wider geographic host range rather than local monophagy? To our knowledge, these questions have not been tested. What else could we test to examine the evolutionary potential for host range expansion? If a very low proportion of larvae survive to adulthood on a non-target plant, compared to the target weed, it seems likely that a population of the candidate agent would not be a threat to the non-target plant in the field. However, could post-release selection result in a higher proportion of larvae that are capable of maturing on non-target hosts? Should we host-range test the offspring of individuals that survived on non-target hosts to determine the potential for the evolution of improved performance on non-target hosts? How many generations should be considered sufficient? Another issue is whether oligophagous weed biocontrol agents are more likely to expand their host ranges after release compared with tightly evolutionarily constrained monophagous species. Where target weeds have no valued close taxonomic relatives in New Zealand, we have used oligophagous biocontrol agents rather than strictly monophagous species. Indeed, if the target weed has close alien relatives that are current or potential weeds, we have deliberately utilised non-target damage. The release of these 'multi-targeting' biocontrol agents is intended to attack both the primary target weed and a range of closely related alien plant species that might currently be minor weeds or perhaps not even have naturalised yet. In some cases, this might include ornamental plants, particularly if they are not widely used or could be readily protected from released biocontrol agents. This 'multi-targeting' is proactive and economically prudent, but is it safe? We end this section with the one example we know where post-release host range expansion is intended. Multiple blackberry rust strains were released in Australia, so that adaptation to different subspecific host taxa will occur (Morin et al. 2006). The risk assessment assumption here is that adaptation will remain constrained within the Rubus fruticosus L. aggregate and not expand onto indigenous Rubus spp. Time will tell whether such a strategy is wise. Recently, the debate over the safety of weed biocontrol agents has moved from direct effects on non-target plants to indirect non-target effects, for example, via interactions in food webs. A high profile example has been the increase in abundance of deer mice Peromyscus maniculatus (Wagner) in rangeland in the USA from feeding on the introduced knapweed gall fly Agapeta zoegana (L.) (Pearson & Callaway 2008). Higher deer mice populations can then cause negative effects on native plants by destroying higher proportions of their seed. Furthermore, as deer mice are the main natural reservoir of hantavirus, the levels of this virus were shown to increase, with potential impacts on humans in which the virus causes a rare but serious disease (Pearson & Callaway 2006). In an entomological example, an introduced tephritid seed fly Mesoclanis polana (Munro) appeared to harm a native insect community by causing local increases in shared native natural enemies (Carvalheiro et al. 2008). More such examples are needed to give a better basis for generalisations. For example, we do not know whether the deer mice–gall fly interaction is unusual, although the potential link to human health seems likely to be. From here on, we concentrate on food webs, but other non-trophic interactions (e.g. direct competition, pollination or other mutualisms) could contribute to indirect non-target effects. First, we look at practical and theoretical food web science and ask how this might be relevant to predicting indirect non-target effects from weed biocontrol agents. Then, we move onto issues of scale of effects and uncertainty in the context of risk assessment. In a review of insect biocontrol, Hawkins et al. (1999) concluded that biological control successes were most often characterised by artificially simplified food webs, typical of productive sector systems (where biocontrol was more successful per se), in contrast to 'natural control' that resulted from multiple links in complex food webs. Recently, biocontrol of weeds has moved from a focus on productive sector weeds to weeds in the natural environment. Does this suggest that biological control of weeds in more complex natural environments is likely to be even less successful than it has been against productive sector weeds, because agents are subjected to 'multiple links in more complex food webs' (Hawkins et al. 1999)? Would we expect biocontrol agents released against environmental weeds to cause more indirect non-target effects than those released against economic weeds? Agents released against environmental weeds are likely to be in closer proximity to native biota and may well have more food web links with this biota. If success rates are also reduced, then there may be more agent species that fail to suppress the target weed but remain reasonably abundant (Pearson & Callaway 2005). However, we are unsure whether a greater number of food web links will necessarily reduce the effectiveness of weed biocontrol agents. For example, insect herbivores do not necessarily show reduced population densities when attacked by more species of natural enemies, provided intra-guild predators are also present (Denno & Finke 2006). Moreover, interspecific plant competition can enhance the impact of biocontrol (Sheppard 1996), and environmental weeds appear to be easier targets for biocontrol than agricultural weeds (Thomas & Reid 2007). Could it be that lower herbivore impacts against environmental weeds are sufficient to achieve weed suppression because these weeds are normally subjected to greater interspecific competition than weeds invading regularly disturbed productive sector systems? We also note that complex food webs have now been linked to community stability in contrast to the previous argument that complexity caused instability (Polis 1998). If so, then the perturbation caused by introducing a biocontrol agent into a complex food web (to blunt an environmental weed invasion) might be expected to create many minor ripples in the web as opposed to fewer major indirect effects. Overall, this probably concurs with the view of a weed biocontrol practitioner (Harris 1988) that in successful biocontrol 'the weed is starting to be cycled through various food chains, a sign of ecological health'. A related issue is whether the strength of a food web perturbation would generally decline with the increasing number of links away from the direct interaction between an introduced biocontrol agent and its host plant. We suspect this would usually be true unless a trophic cascade was set off by impacting a keystone component of a web. A key question then is whether ecological science can help us identify when biocontrol agents might impact on such keystone components of a food web and adjust the risk assessment accordingly. A vital issue is how to avoid introducing agents that become common but still fail to suppress their target weed. Historically, most insect or pathogen species established for biological control of exotic weeds have failed to impact on their targets. Much effort has been invested in modelling to improve agent climate match and to target the vulnerable life stages of a plant, with arguably limited benefit on biocontrol success. Biotic interference reducing the impact of a biocontrol agent because of itself being parasitised or preyed upon, or interacting unfavourably with epiphytic or endophytic microbiota, has been neglected as a factor that influences biocontrol success. Recent studies of these complex biotic interactions indicate that the likelihood of parasitism (e.g. Paynter et al. 2010) and even the structure of a food web (Veldtman et al. 2011) can be predicted prior to an agent's release. More work is required to determine whether the likelihood of predation or disease can similarly be predicted and, if so, what the population level impacts are likely to be. There certainly needs to be an attempt to use food webs as a predictive tool for indirect non-target effects in weed biocontrol (Memmott 2009) and then test these predictions once agents are established: both areas where ecologists could assist the practical discipline of weed biocontrol. The magnitude, and the spatial/temporal extent, of indirect non-target effects is critical for risk assessment in biocontrol. From the above-mentioned discussion of food webs, it is clear that some indirect non-target effects are expected from introducing weed biocontrol, and these will range from positive on some indigenous biota to negative on others. The timing and duration of indirect non-target effects need to be considered in any risk assessment. Do they impact on the valued non-target species throughout the season? Are they likely to decline over time, for example, if the biocontrol programmes become successful (because of either the agent concerned or the addition of further agent species)? With the knapweed gall fly example, it appears that spotted knapweed Centaurea stoebe L. may finally be coming under successful biological control in some areas (e.g. Story et al. 2008), which should prevent the unwanted increases in deer mice populations (Van Driesche, Hoddle & Center 2008). More case studies of indirect non-target impacts of introduced insects and pathogens as weed biocontrol agents are probably needed before valuable generalisations emerge. Whether microbial or insect focussed, we urge that future case studies take a holistic approach to risk assessment, considering spatial and temporal scales as well as the straightforward magnitude of negative (or positive) effects. Overall of course, risk assessment needs to consider the impact of the status quo with the invasive weed. What is needed is a more holistic view – more of an environmental balance sheet. For example, Pearson & Callaway (2008) acknowledge that the indirect effects of the introduced knapweed gall fly on native forb seedlings (from higher populations of deer mice) were dwarfed by the direct negative effects of herbicide used to control spotted knapweed C. stoebe. In another example, a highly host plant–specific weed biocontrol agent, recently introduced into Australia to control bitou bush Chrysanthemoides monilifera (L.) Nordlindh, is associated with declines of local insect communities (Carvalheiro et al. 2008). As the agent shares natural enemies (predators and parasitoids) with seed herbivore species from native plants, this study strongly implicated locally significant apparent competition causing negative effects on indigenous seed feeding insects. However, for risk assessment, we need to know whether this was localised within a few metres of invading bitou bush or more widespread throughout the natural range of the impacted indigenous insects? Work on direct non-target attack has shown that spillover effects can be quite localised (e.g. Taylor et al. 2007) – is the same true for food web effects? If enough ecological studies have been carried out, then we should be informed about the current/potential impact of the weed and know that doing nothing is an option that will result in strong negative effects. Alternatives to biocontrol always exist of course, but in our experience, investment in weed biocontrol is seldom chosen unless these alternatives are expensive or involve unacceptable non-target damage. We hope that the above-mentioned discussion demonstrates many of the areas where the practical discipline of weed biocontrol could benefit from appropriate ecological science. There are relatively few biocontrol practitioners in the world, and often political/logistical/funding constraints to funding ecological science within operational weed biocontrol programmes. However, as mentioned almost ad nauseum in past reviews, biological control systems offer remarkable experimental systems for ecologists. On the positive side, biocontrol practitioners can forge links between the academic end of ecology and on-the-ground stakeholders, and there are areas where the melange of pure and applied science has worked well, for example, release strategies and establishment success. We want to see more such mixing – ecological science should not be the icing on the applied weed biocontrol cake; we prefer a layered Black Forest gateau approach and believe there is an abundance of fascinating ecological research to be undertaken alongside weed biocontrol programmes. Lynley Hayes, Matt McGlone, John Hoffmann, Phil Hulme and an anonymous reviewer improved the manuscript. Funding was provided by FRST contract C09X0905. Simon Fowler and Quentin Paynter are entomologists with over 20-year experience in weed biocontrol in the UK, Australia, USA and New Zealand (NZ). Sarah Dodd has 19-year experience in plant pathology and biological control. Ronny Groenteman obtained her PhD in NZ in 2008, studying multi-targeting biocontrol agents. The authors have experience with classical biological control using >50 agent species on >20 weeds affecting both the productive sector and the natural environment in temperate and tropical regions.

Armillaria species cause economic losses in both pine forests and kiwifruit orchards in New Zealand. In this study Armillaria species-specific PCR primers were developed to facilitate fast and accurate identification of the species found in New Zealand. The first step was to determine which species were present in New Zealand and therefore required distinguishing. Nationally collected Armillaria cultures and specimens had their ribosomal ITS genes sequenced to identify them to species. Sequence data results were consistent with just the four previously recognised species — Armillaria novaezelandiae, A. limonea, A. hinnulea and one as yet unnamed species — being present in New Zealand. Species-specific PCR primers for these four species were subsequently designed targeting the ITS gene region. Specificity of each of the four primer sets for their respective species was confirmed when they were further tested on DNA of the four Armillaria species and other fungi likely to be found in kiwifruit orchards or associated with forest trees.

A project is underway to confirm directly the spread of Armillaria into pine plantations by means of basidiospores.Basidiospores of Armillaria novae-zelandiae were trapped from the air using a SporeWatch volumetric spore trap.Between May and August 2005 the spore trap was placed within an undisturbed podocarp-hardwood forest adjacent to a dense cluster of fruitbodies of A. novae-zelandiae on a fallen tree.Trapping was repeated the following year in an open ar ea close to the margin of the same forest.A molecular identification technique was utilised to detect the presence of A. novaezelandiae basidiospores caught by the spore trap.The technique successfully detected and recorded A. novae-zelandiae basidiospores for a period of weeks during 2005.In 2006 basidiospores were detected for 5 weeks of the testing period at lower quantities, implying filtering of some spores by the tree canopy during dispersal from a more distant spore source.The project is continuing, and this season the spore trap has been placed in the open approximately km from the forest boundary to further test the sensitivity of the technique.Trapping will ultimately be conducted in a pine forest to verify the dispersal of the disease agent by this means.

A project is underway to confirm directly the spread of Armillaria into pine plantations by means of basidiospores.Basidiospores of Armillaria novae-zelandiae were trapped from the air using a SporeWatch volumetric spore trap.Between May and August 2005 the spore trap was placed within an undisturbed podocarp-hardwood forest adjacent to a dense cluster of fruitbodies of A. novae-zelandiae on a fallen tree.Trapping was repeated the following year in an open ar ea close to the margin of the same forest.A molecular identification technique was utilised to detect the presence of A. novaezelandiae basidiospores caught by the spore trap.The technique successfully detected and recorded A. novae-zelandiae basidiospores for a period of weeks during 2005.In 2006 basidiospores were detected for 5 weeks of the testing period at lower quantities, implying filtering of some spores by the tree canopy during dispersal from a more distant spore source.The project is continuing, and this season the spore trap has been placed in the open approximately km from the forest boundary to further test the sensitivity of the technique.Trapping will ultimately be conducted in a pine forest to verify the dispersal of the disease agent by this means.

In 2003, MAF declared a vegetation control zone in Hamilton to limit the potential spread of the Asian gypsy moth.The control zone restricted the movement of vegetation from inside the specified area.This study was designed to investigate the effectiveness of a vegetation control zone as a tool for biosecurity incursions.A preliminary round of interviews was conducted with members of gardening clubs to gather their views.Awareness of the zone and its boundaries was low.Only a few of the interviewees had read material on the vegetation control zone or had noticed road signs on the zone boundaries.With one exception, no members of the groups interviewed had changed their behaviour in response to the zone.It was concluded that understanding public engagement with biosecurity incursion response tools is critical to ensuring their effectiveness.The next stage of the project will explore people's engagement with biosecurity through more case studies.

This paper describes the identification and utilisation of a sequence-characterised amplified region (SCAR) marker specific for the Trichoderma virens biocontrol isolate GV4. The marker was developed from a RAPD-PCR amplification product unique to isolate GV4. When used as a hybridisation probe in Southern blot analysis, it hybridised to the DNA of the species T. virens alone and not to that of other Trichoderma species or closely related genera Gliocladium and Verticillium. The marker also produced a GV4-specific RFLP, distinguishing it from other T. virens isolates when probed to blots with HindII, BamHI or PstI genomic DNA digests. Primers designed from the sequence of the RAPD marker produced a diagnostic amplification product of 346bp for GV4 alone, distinguishing it from all other test isolates. With the exception of one, test isolates did not produce an amplification product with the SCAR primers. The exception was a single Verticillium psalliotae isolate (ICMP5509) that produced a product of approx. 400bp that was easily distinguished from the 346bp product of GV4. The reliability of the SCAR-based diagnostic test was further improved with the introduction of a positive PCR reaction control to each test, achieved by converting the test to a duplex PCR system. Two universal primers flanking the two ITS and the 5.8S region of the ribosomal gene complex were introduced to each reaction to provide a test for PCR reaction inhibitors to eliminate false negatives in the diagnosis. Amplification of this multi-copy genomic region did not reduce diagnostic sensitivity of the single copy SCAR marker. To further increase the sensitivity of detecting GV4 propagules while maintaining a fast sample assessment assay, soil was amended with cornmeal, as a nutrient source, and a mix of antibiotics to favour Trichoderma growth. The soil mix was subsequently incubated for 5 d before total DNA was extracted. Under these conditions, the duplex soil PCR assay detected GV4 down to a concentration of 10 sporesg−1 soil in non-sterile agricultural field soil. This study is the first to report the use of a duplex-PCR diagnostic bioassay for a species within the Hypocrea/Trichoderma genus.

The biocontrol isolate Trichoderma atroviride M1057 and a transformed hygromycin resistant biotype (M1057hygR) were compared using biological control rhizosphere competence and antibiosis studies to determine whether the transformed biotype performed in a similar manner to the wildtype strain In an onion growth chamber trial using soil naturally infested with the onion white rot pathogen Sclerotium cepivorum there was no significant difference (P>005) in the level of disease control given by the two T atroviride strains Similarly populations of T atroviride M1057 and M1057hygR were equivalent (P>005) in the rhizosphere of onion seedlings There was no significant difference (P>005) between the mycelial growth rates of S cepivorum when grown on agar amended with culture filtrate of T atroviride M1057 and M1057hygR Thus T atroviride M1057hygR has similar biological attributes to the wildtype isolate and can be used in future field studies looking at the population ecology of the biological control agent

An isolate-specific restriction fragment length polymorphism ( RFLP) marker previously found for the Trichoderma atroviride ( formerly T. harzianum) isolate C65, an isolate with biological control activity against the kiwifruit stem-end rot pathogen Botrytis cinerea, was modified into a dot blot assay to facilitate the screening of large numbers of leaf and flower/fruit samples for the presence of C65. To increase sensitivity, the dot-blot assay was used in conjunction with a Trichoderma semi-selective medium. This modified diagnostic assay was used to track the survival and spread of C65 on kiwifruit leaves in the shadehouse and flowers/fruit in the orchard over two consecutive growing seasons in the Canterbury region of New Zealand. Results showed that isolate C65 could survive on both leaves and flowers/fruit over an entire growing season. The fungus, applied once in early summer ( late November/early December) to coincide with bud burst, was detected on both leaves and fruit through to harvest in late summer ( March). In addition to its ability to survive, isolate C65 was shown to spread to uninoculated leaves and fruit on the same plant and plants at least 3 m away. It is postulated that the high population of thrips present in the orchard at flowering was responsible for spread of the fungus within the orchard and that resident insects or wind currents could be responsible for spread in the shadehouse. The ability of C65 to survive and spread in the phylloplane and fructoplane of kiwifruit vines over an entire growing season makes it an ideal candidate biological control agent for reducing B. cinerea inoculum in the orchard at harvest and, consequently, post-harvest fruit rot.

Previous work identified an Ulocladium atrum (LU850) isolate with potential as biocontrol against Botrytis on grape (Vitis vinifera). The aim of this study was to find a molecular marker that would distinguish the biocontrol isolate from other isolates of the same species and closely related genera to enable subsequent monitoring of its survival, establishment, and spread in the field. DNA of 53 fungal isolates representing the three genera Ulocladium, Alternaria, and Pithomyces were screened with 10 universally primed polymerase chain reaction (UP-PCR) primers and 28 random amplified polymorphic DNA (RAPD-PCR) primers to screen for bands unique to the biocontrol isolate. Three RAPD-PCR primers produced a single band unique to isolate LU850. However, resultant band patterns and subsequent more detailed morphological studies revealed the biocontrol isolate to be Pithomyces chartarum and not U. atrum. Since some isolates of P. chartarum produce the toxin sporidesmin, which is responsible for facial eczema in cattle and sheep, the toxin producing status of isolate LU850 will need to be determined before it can be considered for further development as a biological control agent.

The volatile fungal metabolite 6-pentyl-α-pyrone (6PAP) suppressed Athelia rolfsii disease of lentil seeds and seedlings when applied onto the surface of the soil, on 13 mm diameter Whatman assay disks. The metabolite had no phytotoxic effects on lentil. This is the first study to show that 6PAP is active in the soil against the vegetable pathogen A. rolfsii.

Eight test organisms were assessed in the glasshouse for their ability to suppress damping-Off of beetroot, induced by isolates of Pythium debaryanum or P. ultimum. Test organisms were applied as seed treatments (10(6) propagules/ml in 2% methylcellulose) and the seed was sown in steam-sterilised seedling mix, which had been artificially inoculated with the respective Pythium spp. A comparison was made with a fungicide (captan) seed treatment. The number of emerged seedlings was recorded at 2 weeks and the number of healthy seedlings and seedling dryweight were recorded at 4 weeks. Four of the test organisms (Penicillium raistrickii, P. simplicissimum, Penicillium sp.(a), and Pseudomonas fluorescens ) gave a consistently high level of disease control for each Pythium spp. at both 2 and 4 weeks. This is the first record of biocontrol activity exhibited by Penicillium raistrickii and P. simplicissimum. In particular, P simplicissimum gave complete control of damping-off induced by P. debaryanum and produced a seedling biomass value equal to that of the uninoculated control. In addition, an isolate of Streptomyces gave complete control of damping-off reduced by P ultimum but faded to control P. debaryanum. These five isolates are considered to be the most promising test organisms for further evaluation.