The science, art and business of successful bioherbicides

Biological Control 52 (2010) 230–240

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Review

The science, art and business of successful bioherbicides G.J. Ash * E.H. Graham Centre for Agricultural Innovation Charles Sturt University & Industry and Investment NSW, Boorooma Street, P.O. Box 588, Wagga Wagga, NSW 2650, Australia

a r t i c l e

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Article history: Received 13 February 2009 Accepted 19 August 2009 Available online 28 August 2009 Keywords: Biopesticide Mycoherbicides Genetic engineering Formulation Fermentation Commercialization

a b s t r a c t In this article, the critical issues in the production of successful bioherbicides have been defined in an effort to stimulate discussion on the underlying science, art of formulation and fermentation and the business of producing and marketing of bioherbicides. To a large extent the science of bioherbicides has focussed on epidemiology, although the enormous potential of molecular technology to improve the efficacy of these agents is being investigated. Some of this potential is coming to fruition in terms of development of tools for the identification and tracking of biological controls, although the genetic modification of biological control agents is still in its infancy. On the other hand, knowledge in the areas of formulation and fermentation is often proprietary in nature. This makes it critical for researchers to work in collaboration with other researchers or industry in these areas. The importance of the appropriate involvement of industry and commercialization partners early in the development process should not be underestimated. Although ad hoc research into biological control should not be discouraged, researchers should be encouraged to think carefully before they postulate on the potential of a bioherbicide based purely on preliminary isolation and pathogenicity testing. As much of the research is specific to a single pathogen/host system, the way ahead in bioherbicide research would appear to be the development of consortia or research nodes in which scientists and business people with backgrounds in the discovery, development and commercialization of biopesticides work collaboratively on a number of projects. There has been movement towards this type of model in countries such as Canada, USA and New Zealand although other countries lag behind. Interestingly, all five of the recently registered bioherbicides in the U.S. and Canada were developed and registered by small-business enterprises or a subsidiary of enterprises with no prior record in pesticide development. The constraints to bioherbicides are not in the science, art or business: it is in bringing all of these aspects together in an accessible way and the sharing of intellectual property in an equitable fashion. A new model for the commercialization of bioherbicides should build on currently established research networks, but need to have a stronger link to industry (especially small–medium enterprises) and requires funding for infrastructure and personnel. This funding needs to come from the public sector. Industry is interested in engaging this type of research, but they need to be reassured that the approach is feasible, economic and realistic and that the resources required are available. Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.

1. Introduction Bioherbicides are a subset of biopesticides which have been variously described, but in the context of this article, will be restricted to microbially-based pesticides for the management of weeds. This definition is similar to the one used by Crump et al. (1999) which varies from that of Hoagland (1990) in that it excludes phytotoxins or secondary metabolites. In a majority of cases the bioherbicide includes a fungal organism as the active ingredient, although there are a few examples of the use of bacteria (Anderson and Gardner, 1999; Daigle et al., 2002; DeValerio and Charudattan, 1999;

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Imaizumi et al., 1997; Weissmann et al., 2003) and viruses being used or proposed to be used as bioherbicides (Charudattan and Hiebert, 2007; Ferrell et al., 2008). In addition to the active ingredient these microbes are routinely formulated in various ways to improve their delivery and efficacy. 1.1. Defining the success of bioherbicides If we are to define success in biopesticide research as the registration and commercialization of a product (Rosskopf, 2007), then the success rate in this area has been low. Since the early successes of the use of Phytophthora palmivora and Colletotricum gloeosporioides f.sp. aeschynomene as bioherbicides (Ridings 1986; (Tebeest et al., 1992), there have been at least 15 products commercialized.

1049-9644/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2009.08.007

G.J. Ash / Biological Control 52 (2010) 230–240

Charudattan (2001) reported eight successfully registered or commercially available bioherbicides, the majority of which were fungal based, with a further three bioherbicides registered in Canada since 2002 (Bailey et al., 2010). In addition to these commercialized products, Charudattan (2001) also listed over 50 examples of pathogen-weed combinations which had been reported as having potential as bioherbicides. Despite approximately only 11 bioherbicide products being made available in the marketplace, a further search of the literature using the ISI Web of Science1 database has revealed 509 papers published which mention bioherbicides or mycoherbicides (as of February 2009) since 1987. The majority of these papers were from USA (36%), Canada (20%) and Australia (7.8%). During the same period there were over 17,000 papers published which mentioned synthetic herbicides. This reflects, among other things, the differential funding expended in the different areas. Up to 15,000 compounds per year were screened for herbicidal activity for each herbicide that successfully reached the market in the 1980s, for example (Hoagland, 2001). Many authors of papers on bioherbicides appear to try to legitimize their research by reporting the ‘‘potential as a mycoherbicide or bioherbicide” in the title (59 papers since 1987). If this search is extended to the mention of this phrase in the topic there were nearly 335 of the 595 papers which do so. In many cases the research is undertaken by students and there is very little if any attempt at true commercialization of the product. This has led to ‘‘less than spectacular results” (Bailey et al., 2010). Charudattan (2005) states that over 8% of bioherbicides have been verifiable successes, but does go onto acknowledege the low sample numbers may have a large influence when determining success rate. Of course, if the commercialization of a bioherbicide is sought, then patents may be pursued to protect the intellectual property and to help attract investment in the project. Using Patent Lens2 a total of 71 patents were found which mentioned bioherbicides in their titles (USA (59); Europe (9); Australia (3)). Interestingly, none of the three granted patents in Australia were from Australian scientists. In the past the linear or scientific model has been employed by researchers in the development of bioherbicides. In the majority of cases a target pathosystem is chosen based on the perceived need for a biological alternative within the system by the researcher or an industry member. Often the research tends to begin with experimentation on activity and host range testing in the glasshouse and studies on epidemiology to define the optimum conditions for infection. Small scale field experiments are then used to demonstrate the potential of the biocontrol agent in ‘‘real world” situations. To produce sufficient inoculum, small scale fermentation is undertaken to produce spores or mycelium and the testing of different formulations and adjuvants is required. At this stage the biocontrol agent is often presented to a business partner as a fait accompli. The research can be classified in to several stages under the identification of opportunities for the development of biological control agents (the drivers), the discovery and genetic improvement of the organism (the science), the fermentation and formulation of the agent (the art) and the commercialization of the product (the business).

2. Opportunities for the development of bioherbicides A number of opportunities for the development of bioherbicides (and biopesticides more generally) have been reported (Charudattan, 2001). In Canada there has been a clear trend for the phasing out of the use of synthetic pesticides in lawns and gardens (Bailey et al., 2010; Cisar, 2004). This has led to a number of research pro-

grams aimed at developing bioherbicides for the management of dandelion (Taraxacum officinale) in turf grass in Canada (AbuDieyeh and Watson, 2007; Bailey and Derby, 2001; Schnick and Boland, 2004; Schnick et al., 2002; Zhou et al., 2004). A commercial product using Sclerotinia minor has received registration from the Health Canada’s Pest Management Regulatory Agency3 and had limited availability in 2008 (Watson, 2008). In this case, the perceived lack of negative environmental impact of bioherbicides has created a market for products left when the synthetic pesticides have been withdrawn. Herbicide resistance can also be seen as a driver for research into bioherbicide development. The research of Ash, Cother and colleagues (Cother, 1999; Cother and Gilbert, 1994a,b; Fox et al., 1999; Jahromi et al., 1998) was supported in part due to the emergence of resistance in the target weed to synthetic pesticides. They demonstrated that the fungus Plectosporium alismatis (sensu (Pitt et al., 2004)) could be used to manage weeds in the family Alismataceae in rice fields in Australia. Jahromi et al. (2006) suggested that synergy between P. alsimatis and selected herbicides could lead to reduced herbicide application rates. Synergy with synthetic herbicides has been seen as a mechanism to overcome resistance in the host and therefore reduce the application rate of both the herbicide and the biological control agent in a number of systems (Boyette et al., 2008; Hoagland, 1996; Peng and Byer, 2005; Schnick et al., 2002). However, the use of reduced rates of synthetic herbicides may be a double edged sword, a fact noted by several authors, as the use of below label rate use of herbicides could contribute to herbicide resistance (Busi et al., 2009; Christoffers, 2009). The combination of a microbial active ingredient and synergy with synthetic pesticides is discussed further under bioherbicide formulation below. Due to the enormous costs involved in the production of synthetic pesticides, global companies have tended to focus on the registration of pesticides in the major crop/cropping systems. This has led to a number of attempts to develop bioherbicides in these non-core or niche markets (Charudattan, 2001). These markets could be of considerable size and include those which have been created by synthetic herbicide withdrawal and the organic food movement. The use of mycoherbicides is compatible with the philosophy of organic food production, provided the agent had not been genetically modified, the carriers and adjuvants are natural products and the host range is not considered to be too wide (Rosskopf and Koenig, 2003). In terms of land area dedicated to organic food production, growth rates of over 20% per annum have been reported in many western countries (Bruinsma et al., 2003), although these figures need to be treated with some caution due to the small starting base levels. This increase is predicted to continue largely due to the continued demand for organic foods and the subsidization of this type of agriculture by many governments (Bruinsma et al., 2003). This market for bioherbicides might be larger than first predicted due to the localization and even globalization of organic food (Clarke et al., 2008). Hallett (2005) suggested that parasitic weeds could offer a niche system for the implementation of bioherbicides as parasitic weeds are not adequately controlled through the use of herbicides or traditional weed management strategies (Aly, 2007). As an example of this approach, dodder species (Cuscuta spp.) have been targeted using the fungal pathogen Alternaria destruens (Bewick et al., 2000). Two bioherbicides based on this pathogen (strain 059) have been registered in the USA; Smolder G and Smolder WP (EPA Reg. Nos. 34704-824 and 34704-825)4. Additionally, Lubao II, a formulation of Colletotrichum gloeosporides f.sp. cuscutae, 3

1 2

http://apps.isiknowledge.com/. http://www.patentlens.net/daisy/patentlens/patentlens.html.

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http://www.pmra-arla.gc.ca/. http://www.ep a.gov/opp00001/biopesticides/ingredients/factsheets/ factsheet_028301.htm. 4

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has been used in China for dodder control (Zhang, 1985). Considerable research has also been undertaken on the biological control of striga (Striga spp.) (Elzein et al., 2006; Sauerborn et al., 1996); and broomrapes (Orobanche spp.) (Aly, 2007; Boari and Vurro, 2004; Elzein et al., 2006; Ghannam et al., 2007; Hallett, 2005; Müller-Stöver et al., 2004; Sauerborn et al., 1996; Zermane et al., 2007). In addition to these biological/social drivers for the production and adoption of bioherbicides, there are changing economic drivers. The increase in the cost of farm inputs and the price for farm products in recent years will also have an impact on bioherbicide research. It maybe expected that farmers will be prepared to pay a premium for inputs such as biopesticides and this is likely to affect the types of approaches used to produce new biopesticide products in some markets. 3. The science of bioherbicides Essentially the discovery and development of bioherbicides has been undertaken by plant pathologists, weed scientists or entomologists and begins with the observation of naturally occurring diseased plants. The early stages of the investigation follow traditional plant pathology techniques, including the application of Koch’s postulates and other standard techniques taught in undergraduate plant pathology (Agrios, 2005). At this stage high concentrations of mixed inoculum are used to increase the likelihood of plant mortality. Following the isolation of a likely candidate, the research program often includes culture purification, host range testing, laboratory scale fermentation and preliminary formulation. A number of reviews have offered reasons why bioherbicides developed in this way have not been successful (Auld and Morin, 1995; Hallett, 2005; Weston, 1999). Some authors have suggested that the application of molecular biology to bioherbicides may overcome some of these shortcomings in bioherbicides (Amsellem et al., 2002; Gressel et al., 2005). As the techniques and information available through molecular biology is changing so rapidly, this will be the focus of this section on the science of bioherbicides. Further, much of this information, although focused on bioherbicides, is directly relevant to classical biological control, especially the sections on pathogen and host diversity and tracking of biological control agents. 3.1. New directions in the application of molecular biology to bioherbicide research The major advances in nucleic acid techniques afford many opportunities to understand and improve inundative biological control of weeds. Presently, the research concerning the biological control of weeds using bioherbicides has been ‘‘low-tech” with a predominately discovery and epidemiological approach to the development of products and an agronomic approach to their application. There are a number of ways in which the application of molecular biology could help to improve the success of bioherbicides. 3.2. Genetic diversity of the host Genetic diversity studies of the host or target species is becoming common place. This is often important so that a representative population of the target can be chosen in pathogenicity and host range testing. In biotrophic relationships there is evidence for race and formae specialis variation which may impact on the effectiveness of biological control. This is more important in classical biological control as compared with the bioherbicides approach as biotrophs are seldom used as bioherbicides. However, in the case of hemibiotrophs and some necrotrophs there is greater evidence

for variation between isolates and resistance in the host (Antonio et al., 2008; Delourme et al., 2008; Wunderlich et al., 2008). There is an ever increasing range of examples of the assessment of the genetic diversity of the target weeds in bioherbicides programs using a range of marker and molecular approaches. (Ash et al., 2004, 2003; Okoli et al., 1997), The utility of DNA markers can be assessed in terms of their informativeness (a measure of their polymorphic information content), the multiplex ratio (the number of loci which can be assayed simultaneously) and their genotyping error (a reflection of reproducibility and clarity). The types of markers that are commonly used include Restriction Fragment Length Polymorphisms (RFLP), Random Amplified Polymorphic DNA detection (RAPD), Simple Sequence Repeats (SSR), Amplified Fragment Length Polymorphisms (AFLP), Single Nucleotide Polymorphisms (SNPs) and Diversity Array Technology (DArT). These types of markers vary in their reproducibility, level of polymorphism detected and cost. Ultimately the selection of the type of marker used is dependent on the aims of the study. A discussion of the types of markers available and their strengths and weaknesses is beyond the scope of this review, but readers are directed to Sunnucks (2000) and Yang et al. (2006). In the future, these studies should be used to direct pathogenicity studies so that the full diversity of the weed population can be targeted, and will hopefully lead to greater field efficiency and reduced variability in management of weeds through the use of bioherbicides. 3.3. Genetic diversity of pathogens A corollary to the study of genetic diversity of the host is the study of genetic diversity of the pathogen. An understanding of the genetic diversity of the organism involved as the active ingredient in a bioherbicide is important for a number of reasons. As many of the agents have ambiguous morphological characters, the use of genetic fingerprinting and sequencing can assist in the identification of the organism. In fungi, these molecular traits are often based on the sequence variation in the internal transcribed sequence (ITS) region of the rDNA, the b-tubulin gene or the elongation factor a gene. These sequences have been used by a number of authors to characterize the pathogen of interest (Berthier et al., 1996; Tessmann et al., 2001; Yourman and Luster, 2004). Information from these regions varies in its usefulness based on the degree of variation and the number of sequences currently available on the database for the organism in question. Ash et al. (2009) used a combination of fingerprinting and sequencing to postulate that the isolates of Phomopsis that they had isolated from Carthamus lanatus were distinct from those found to be pathogens of crops. This type of information may be used in supporting packages for the registration of bioherbicides. It is also important when dealing with pathogens with potentially wide host ranges, such as Phomopsis. Further, when used in conjunction with studies on the genetic diversity of the host or target weed it may help in predicting suitability of weed/pathogen systems for biological control by either inundative or classical methods (Charudattan, 2001). Fingerprinting and sequencing techniques can also be used to track and quantify the level of the bioherbicides in the environment. Zhou et al. (2004) demonstrated the monitoring of pathogen movement and persistence in soil using traditional, specific PCR. They found that the pathogen they were using, Phoma macrostoma, had limited dispersal and longevity in the environment. This, they suggested, would indicate that the pathogen would have limited environmental effects, and thus would be easier to register as part of a bioherbicide. Zhou et al. (2004) also suggested that quantitative PCR would improve the sensitivity and utility of their assay. In 2006, Dauch et al. (2006) demonstrated the utility of real time, quantitative PCR for the quantification of Colletotricum cocco-

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ides in planta. This approach could be used to detect and quantify this pathogen to determine its environmental fate. As the cost of sequencing continues to fall, it will become more commonplace for representative isolates to be sequenced (whole genome). This will facilitate the development of specific markers for environmental tracking, quantification, identification, gene discovery and protection of intellectual property in bioherbicides. 3.4. Increasing genetic diversity in biocontrol agents Mutation of microbial strains has been used as a means of increasing production of various enzymes and industrial products from fungi and bacteria. However the mutagenesis of biopesticides is not as common. Butters et al. (2003) analyzed naturally occurring fungicide mutants of Beauveria bassiana using degenerate primers and were able to identify nine sites which conferred tolerance to methyl benzimidazol-2-yl carbamate (a fungicide). In bioherbicide research, Hoagland et al. (2007a) found decreases in virulence in the biocontrol fungus Myrothecium verrucaria from isolates taken from morphological sectors on agar. Some of the mutants where just as efficient as a biocontrol agent as the wild type, however, none were more virulent. Hoagland et al. (2007a) also indicated that they would use the approach to search for mutants with low mycotoxin production. Polyploidy is thought to be common in plants, with the incidence ranging from 30% to 80% (Masterson, 1994) and is also widespread in fungi (Deacon, 2006). Polyploidy often gives rise to cascades of novel gene expression patterns in plants. This invariably leads to new phenotypic variation. In fungi, polyploids, aneuploids and heterokaryons occur. The effects of polyploidy in fungi are not well understood, but, based on what occurs in plants, could lead to over expression of genes, silencing of others or completely unpredicted outcomes. In yeast for example, levels of ploidy have been shown to affect the expression and the susceptibility to virally-mediated toxin (McBride et al., 2008). At the time of printing, there was no evidence of published material on the improvement of bioherbicides using increased or altered ploidy levels. As an alternative strategy to the use of protoplast fusion, it would seem that the investigation of changes in virulence due to changes in ploidy may be an interesting avenue of research. The fusion of protoplasts is viewed as an alternative approach to increasing the genetic diversity of pathogens and has been attempted in various fungi and actinobacteria (Agbessi et al., 2003; Aiuchi et al., 2007; Balasubramanian and Lalithakumari, 2008). Zhang et al. (2007) used the fusion of protoplasts of Helminthosporium graminearum subspecies echinochloe with Curvularia lunata to complement the pathogenicity and spore production abilities of the two fungi. They successfully demonstrated the fusion of the isolates using PCR, but suggested that the majority of the DNA arose from the Helminthosporium isolate. Some of the resultant fusants had increased sporulation and production of the phytotoxin ophiobolin A. This toxin, present in a crude extract, adequately controlled Cyperus difformis in the field. This then is not truly a bioherbicide using the definition within this paper. It does however introduce the improvement of bioherbicide candidates without the use of molecular techniques. This could also include the improvement of virulence through mutagenesis or an increase in ploidy levels within the pathogen. The production of fusion mutants through the use of protoplast fusion, under the definition of genetic modification used by the Office of Gene Technology in Australia5, would be considered genetic modification and so would be regulated under the Gene Technology Act 20006 in Australia, and 5

http://www.ogtr.gov.au/. http://www.frli.gov.au/ComLaw/Legislation/ActCompilation1.nsf/current/bytitle/ BCD8BD745138702DCA257313000D87D5?OpenDocument&mostrecent=1. 6

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under similar legislation within other countries. Presumably this type of modification would not be able to be used in the European Union, due its embargo on GM crops and products.

3.5. Genetic modification of the pathogen Since Dickman et al. (1989) demonstrated that the host range of a pathogen could be modified with the insertion of a single gene involved in the infection process, genetic modification of fungi has been touted as a direction of the future to improve the virulence of bioherbicidal organisms and also to reduce the reliance of the organisms on the environment during the infection process (Amsellem et al., 2002; Gressel et al., 2005). The modification or insertion of enzymes of fungal pathogens involved in plant penetration, such as cell wall degrading enzymes and cutinases, does not always lead to increased virulence due to the considerable genetic redundancy in these genes (Xu et al., 2006). Cohen et al. (2002) reported the increased virulence of Fusarium oxysporum using up-regulation of indole acetic acid production. This increase in virulence was not considered sufficient to warrant the use of this transformation. However, Amsellem et al. (2002) reported a ninefold increase in virulence and a reduced requirement for moisture in C. coccoides, a bioherbicidal candidate against Abuliton theophrasti, when using the NEP1 gene from F. oxysporum. The NEP 1 gene encodes the Nep 1 protein, a potent phytotoxin. These researchers reported that although the gene improved the virulence of the organism it also increased the host range to tomato and tobacco, an undesirable characteristic in this case. However, Dauch et al. (2006) could not reproduce the increased virulence to A. theophrasti when using the C. coccoides strain T2O-a when compared to the wild type strain. Furthermore, they could not detect the gene expression in culture or in planta. They suggested the gene was being silenced. Amsellem et al. (2002) noted that over expression was inhibited within F. oxysporum and hypothesized that this would not be the case in unrelated fungi. However, as the pathogen undergoes a biotrophic phase for a period of up to five days postinoculation, the constitutive expression of a phytotoxin may reduce the biomass of the fungus and could interfere with the plant–pathogen interaction at this stage. This would likely lead to a reduced level of symptom expression. Other genes for a range of polyketide synthetases have been identified in a number of plant pathogenic fungi (Xu et al., 2006) which could also be useful in selectively increasing virulence. Use of these types of virulence factors would require the use of inducible promoters which are aligned to the fungal/plant interaction being targeted. Alternatively, the modification or deletion of genes such as the NADPH oxidase genes implicated in the regulation of symbiosis in some plant/pathogen interactions (Tanaka et al., 2006) could be used to alter the host range of some bioherbicidal agents. Currently available sequence data from non-filamentous fungi such as yeasts or filamentous non-plant pathogenic fungi may not be overly useful in the search for targets for improved virulence as they may not contain homologs of pathogenicity or virulence genes. For example, the two closely related species, Aspergillus fumigates and Aspergillus fischerianus have 9226 homologs with 700 genes with no homology between the two species (Nierman et al., 2005). Of the fungi which have been identified as having potential to be developed as mycoherbicides, only Sclerotinia sclerotiorum and F. oxysporum have been fully sequenced (Xu et al., 2006). The speed of identification of genes involved in plant/pathogen interactions will increase as sequencing information of more plant pathogenic fungal genomes becomes available in the future (Xu et al., 2006). This will increase our knowledge of pathogen virulence and may lead to better understanding of regulation of plant–pathogen interactions in bioherbicides.

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4. The art of bioherbicides 4.1. Formulation of bioherbicides Formulations of biopesticides contain the active ingredient (in this case the microorganism or spore), a carrier (largely inert material) and adjuvants which may contain compounds such as nutrients and/or chemicals which aid in the survival of the pathogen or help in protecting the active ingredient from adverse environmental conditions (Hynes and Boyetchko, 2006). The adjuvants may also aid in the infection of the host. There has been a dearth of detailed information on formulation of bioherbicides in the literature until recently, which could be attributed to the proprietary nature of the research (Fravel, 2005; Hynes and Boyetchko, 2006) or the lack of understanding of the basic microbial processes being affected. As such, the formulation of biopesticides is often viewed as an art rather than a science. Many believe that improvement in formulation of bioherbicides may provide the key elements to increasing success of the agents in the field (Auld and Morin, 1995) and as the formulation may have a great effect on the shelf life of the product, it will also affect the development, registration and commercialization (Ghorbani et al., 2005). The formulation of biocontrol agents is dictated by the type of organism being formulated but ultimately must ensure that the agent is delivered in a form that is viable, virulent and with sufficient inoculum potential to be effective in the field. Furthermore, the formulation may also dictate the means of delivery of the final product, for example as a seed dressing or a foliar applied formulation. To be successful a formulation must be effective, economical and practical to use. Formulation can be divided into dry products (dusts, granules, pellets, wettable powders, encapsulated products) and liquid formulations (suspensions, and emulsions and encapsulated products) (Auld et al., 2003). One of the main constraints to the success of bioherbicides has been attributed to the long dew period required for infection by some pathogens on the aerial surfaces of target weeds (Auld et al., 2003; Auld and Morin, 1995). Since the publication of the original article by Auld and Morin (1995) there has been extensive research on many bioherbicide formulations to reduce the reliance on extended periods of moisture in the prepenetration phase. Simple formulations containing wetting agents such as Tween 20 and the silicone-polyester copolymers (Silwet) enhance leaf wetness and bacterial entry into leaves, and although there has been some reported success with these types of formulations (Auld et al., 1990; Cother and Gilbert, 1994b; Jahromi et al., 2006) they may increase moisture evaporation and thus lead to variability in field control (Auld et al., 2003). In search of improved liquid formulations, researchers have added polymers (Chittick and Auld, 2001; Shabana et al., 1997), vegetable oils in simple emulsions (Auld, 1993; Bourdot et al., 2006b; Boyette et al., 2007a), produced invert emulsions (Boyette et al., 1993; Egley et al., 1993) and so called water-in-oil-in-water (Auld et al., 2003). Some, but not all, of this research has proven successful. None has yet led to the commercialization of the selected bioherbicide. Solid formulations including dusts, granules, pellets, wettable powders and encapsulated products have also been widely researched. These formulations may be applied in a liquid but more often are better suited to broadcast or soil application methods. The formulations are designed to allow survival of the active ingredient during storage while allowing rapid dispersal of the agent under favorable conditions (Auld et al., 2003; Hynes and Boyetchko, 2006). This survival may be enhanced through the use of modified packaging atmospheres and temperatures (Teshler et al., 2007). The formulations may also allow secondary growth and reproduction of the active ingredient during periods of favor-

able climatic conditions (Boyette et al., 2007b) so effectively increasing the inoculum potential of the active ingredient post application. Soil applied formulations should also contain nutrients to permit the organism to survive until it is established in the rhizosphere or spermosphere (Hynes and Boyetchko, 2006). In the case of fungi as the active ingredient, the carrier of the dry formulation is often derived from the solid substrate from which the organism is produced. Cracked grain has been used successfully by a number of researchers (Bourdot et al., 2006a; Schnick et al., 2002; Vogelgsang et al., 1998) as part of the formulation. Alternatively, the organism may be grown in a submerged culture and the mycelium, spores, bacterial cells or other propagules incorporated into a matrix of calcium alginate (Walker and Connick, 1983), flour and kaolin — known as ‘‘Pesta” (Connick et al., 1997, 1992; Müller-Stöver et al., 2002; Shabana et al., 2003), or starch, sugar, corn oil and silica — known as ‘‘Stabileze” (Amsellem et al., 1999; Quimby et al., 1999; Zidack and Quimby, 2002). The particles produced from some of these formulations are often quite large and so present a problem in terms of application rate and coverage. An alternative method which produces particles as small as 50 lm in diameter is microencapsulation (Chittick et al., 2003; Winder et al., 2003). In addition to formulation components outlined above, there are numerous other adjuvants which have been tested for their ability to increase the effect of the bioherbicide by protecting the active ingredient from desiccation and other environmental effects (Bailey et al., 2004; Boyette, 2006; Boyette et al., 1996; Neumann and Boland, 1999; Zhang et al., 2003). Wyss et al. (2004) tested a range of adjuvants commonly used in pesticides for their effect on the mycelia growth and conidial germination of Phomopsis amaranthicola, a biocontrol agent for species of Amaranthus. They hypothesized that bioherbicides would need to be compatible with a range of commonly used pesticides and adjuvants if they are to be commercialized. In most cases, adjuvants were moderately to highly compatible with biological agents. Commonly, researchers will test for some sort of synergistic interaction between the bioherbicidal candidate and synthetic herbicides in an effort to reduce the quantity of these chemicals being used in the cropping system or in an effort to increase the efficacy of the bioherbicide itself (Christy et al., 1993; Graham et al., 2006, 2007; Hoagland, 1996; Jahromi et al., 2006; Peng and Byer, 2005). Many synthetic herbicides are inhibitory to conidial germination of fungi while others are relatively benign (Graham et al., 2006). In addition to the herbicide active ingredient, the formulation of the herbicide may have adverse effects on the active ingredient of the bioherbicide. Incorporation of these compatible herbicides into bioherbicide formulations may decrease their application rate by greater than 10-fold (Jahromi et al., 2006) or give greater control of the target weed than either agent alone (Graham et al., 2007). All of these interactions with pesticides will have an effect on the timing and type of application of the bioherbicide, for example as tank mix or as a sequential application with other pesticides (Wyss et al., 2004). Some fungi produce an extracellular matrix which has been shown to give a level of UV protection to the spore (Mondal and Parbery, 2005). This effect can be mimicked by formulations which can also be used to protect pathogen propagules from UV radiation. Most studies on the effect of UV radiation on fungi have been undertaken on insect pathogens (Moore et al., 1993; Shah et al., 1998). However, in recent studies (Ghajar et al., 2006a,b,c) it has been demonstrated that UVB has a significant effect on conidial germination, appressorial formation and melanization in the two bioherbicidal fungi, P. alismatis and Colletotrichum orbiculare with the latter fungus being the most susceptible to the radiation. Furthermore, they also found that UVB radiation stimulated

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microcycle conidiation (the formation of secondary conidia from germ tubes) in P. alsimatis. On the other hand, they found that short exposure to UVA could promote appressorial formation (Ghajar et al., 2006b). They went onto to test a variety of UV protectants of which propyl gallate and mineral oils were suggested as the most likely to be economically feasible (Ghajar et al., 2006c). In addition to interactions with a range of chemicals, the effect of other biologicals in the formulation or on the plant may alter the efficacy of the bioherbicide (Ditmore et al., 2008; Morin et al., 1993a; Pitelli et al., 1998). Boyette et al. (1979) first demonstrated the use of a combination of pathogens in a bioherbicide. This concept was suggested by Auld et al. (2003) as a means of expanding the market for bioherbicides. This approach has since been used by several research teams (Mitchell et al., 2008; Morin et al., 1993a,b) Chandramohan and Charudattan (2003) successfully controlled three different weed species using a combination of four fungal pathogens in a single mixture in the glasshouse. They demonstrated that there was no loss in efficacy of the individual fungal pathogens when used in a mixture. However, caution in this approach might be advised as there is the possibility of inducing systemic acquired resistance in the host when pathogens are used in combination, which could reduce the efficacy of one or all of the fungal pathogens. If a pathogen induces a response that it subsequently evades it may affect the ability of another pathogen to infect. This has been recently inferred by Ditmore et al. (2008) as a mechanism of interaction between two isolates of C. gloeosporioides f. sp. aeschynomene. In most of the cases outlined however, the formulations have been developed with a small number of active ingredients and have not been developed using general principles which are applicable to a whole class of bioherbicides. This approach was suggested by Hynes and Boyetchko (2006). Hynes and Boyetchko (2006) went onto encourage research linkages with scientists in the pharmaceutical and food industries, rather than the herbicide or pesticide industries as a means of improving formulation for biopesticides as a whole. 4.2. Application technology It is well known that application technology has an important influence on the efficacy of synthetic pesticides, but the understanding of its effect on bioherbicides has been poorly investigated (Doll et al., 2005; Lawrie et al., 2002a,b). Retention of spray droplets can be affected by surface characteristics and morphology of the weed and its biotypes, as well as the adjuvants, travel speed and droplet sizes (Katovich et al., 1996; Ramsdale and Messersmith, 2002; Singh et al., 2002). The findings of Doll et al. (2005) that disease severity caused by the fungus Microsphaeropsis amaranthi on Amarathus spp. is affected by droplet size and coverage are not surprising, but do indicate that more attention needs to be paid to application technology of bioherbicides in the future. Byer et al. (2006) also found a greater efficacy of their Colletrotrichum truncatum with smaller droplet sizes on scentless chamomile (Matricaria perforata). However, droplet size was not important in the efficacy of C. gloeosporides f.sp. malva on round-leaved mallow (Malva pusilla). They attributed this to greater spray retention on the vertical stems in this system. Once again this indicates that the biology of the host/pathogen relationship can subtly affect the efficacy of the bioherbicide. As the active ingredient of a bioherbicide is a living organism, nozzle type, pressure and the types of carriers used may all affect the survival of the organism prior to deposition on the target. 4.3. Fermentation Hocahuser, 1983 (as cited by Dernain (2006)) wrote ‘‘under the most rigorously defined conditions of temperature, pH, aeration,

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and nutrient conditions, the organism will do whatever it damn well pleases”. Thankfully this is no longer held to be true, but it does highlight why fermentation is sometimes still thought of as an art rather than a science by some. In the case of biopesticide production the fermentation of the active ingredient is often closely linked to the formulation used (Hynes and Boyetchko, 2006) as mentioned previously. The main types of fermentation used for microbial pesticides are solid-state or solid substrate, submerged and bi-phasic approaches. Solid-state fermentation is the process of growing microorganisms on a solid material without any free water and is more suitable for the production process involving fungi than bacteria (Bhargav et al., 2008). Solid substrate fermentation is a similar process, however in this form the solid material serves as the nutrient source as well as the supporting material (Pandey et al., 2000). Unfortunately in the literature, these two processes are often used interchangeably. In bioherbicide research, the majority of researchers have used solid substrate fermentation, especially in production of fungal pathogens. A range of substrates have been used in the production of bioherbicides including rice (Boyette et al., 1993; Hoagland et al., 2007a), wheat (Bourdot et al., 1995; Masangkay et al., 2000), barley (Schnick et al., 2002; Teshler et al., 2007; Vogelgsang et al., 1998), maize (Babu et al., 2004) and millet (Grey et al., 1995). In many cases, following colonization, the substrates are ground and applied as a granular formulation. Alternatively, conidiation is induced on the solid substrate (Babu et al., 2004; Yamaguchi, 2007). Conidia produced using solid substrate fermentation are often more stable and resistant to stresses caused by drying than those produced in liquid culture (Holker and Lenz, 2005). Other researchers have used extracts from the colonized grain as a bioherbicide. Hoagland et al. (2007a) used rice grain to grow Myrothecium erraria to control kudzu in a range of crops. They found that extracts from the rice grains were more efficacious than inoculum produced from agar when applied to soil. Solid substrate fermentation can also be used in conjunction with an extruder technology to produce a formulation suitable for field application. Using this approach, Daigle et al. (1998), produced an extension factor (the conversion ratio of the fermentation product to the final extruded product) of between 4 and 1600 (dependent upon the biocontrol agent used). This would have an obvious beneficial effect on the economics of producing these types of inoculum. Submerged or liquid fermentation is the system which is most widely used industrially for the production of a variety of microbial products (Gibbs et al., 2000). This process is ideally suited for bacteria. Jackson et al. (1998) reported the production of 2  1012 cells per milliliter of Xanthomonas campestris pv poae, the active ingredient of a bioherbicide for Poa annua ((Imaizumi et al., 1997; Johnson, 1994) in submerged culture at 30 °C using sucrose or glucose as the sole carbon source. As most of the bacteria under consideration as bioherbicides do not produce endospores, they are variously formulated following fermentation to increase their shelf life (Jackson et al., 1998; Zidack and Quimby, 2002). In the case of filamentous fungi, the production of spores in submerged culture can be problematic (Gibbs et al., 2000). However, some fungi considered as bioherbicide candidates will produce conidia or other forms of propagules in liquid culture and so submerged fermentation is an ideal method for their production (e.g. Colletotricum, Plectosporioum, Fusarium). Fungi are also known to produce secondary propagules in liquid culture such as microsclerotia (Jackson and Schisler, 1995) or chlamydospores from hyphae or from conidia (Ciotola et al., 2000; Cliquet et al., 2004; Cliquet and Zeeshan, 2008; Lanoiselet et al., 2001; Müller-Stöver et al., 2002) which can be variously formulated and applied as a bioherbicide. Optimization of growth conditions for bacterial or fungal growth in submerged culture can be achieved through the use of

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response surface methodology (Bhaskar et al., 2008; Cliquet et al., 2004). This approach is regularly used to develop optimized protocols for substrate composition and concentration, pH and temperature. In many fungi, conidiation is completely suppressed in liquid culture (Smith, 1978). However, this does not preclude the use of submerged fermentation in the production of fungi, as the hyphal biomass can be used as a starter in solid substrate fermentation or in other formulations such as granules or microcapsules (Chittick et al., 2003; Daigle et al., 1998). In the research of Briere et al. (2000) it was found that incorporation of spent liquid medium containing high levels of oxalic acid with mycelium in sodium alginate granules enhanced the virulence of isolates of S. minor against common dandelion.

5. The business of bioherbicides If the gauge of the success of a bioherbicide is its commercialization, then there are many aspects of business and marketing which determine the success or otherwise of the venture. Lidert (2001) asked the question of biopesticide researchers ‘‘if it is so good, why is it so bad”, referring to the oft quoted reasons for the use of biopesticides when compared with their rate of commercial success. He went onto lay much of the blame for this lack of commercial success at the feet of the scientists and the institutions at which they work. He perceived a lack of purpose, in that, the aim of the research was in fact good science and publications rather than bringing a product to market. There are large numbers of examples within the scientific literature which purport the potential of bioherbicides but which have little chance of success (as noted in the introduction to this review). So how does one differentiate novel scientific research from another potential bioherbicide? Pathogen virulence is paramount to the efficacy of the bioherbicide (Charudattan, 1988). Without a virulent pathogen, the likelihood of success is low. However, this must be accompanied by a suitable target, a driver or reason for the product and the ability to produce and market a product that is competitive within the marketplace. Many commercial partners have a wealth of knowledge on markets and product development so their input may help in these very early stages of product development to decide on the likely target and candidate organism. Researchers should also be encouraged to abandon referring to their ‘‘pet pathogen” as a bioherbicide candidate. In many cases, long after they have failed to demonstrate the economic and biological rationale for the production of a bioherbicide the researcher continues to research and publish on the system they have studied for so long. Although these studies add to our knowledge of the pathosystems and are often useful in terms of a retrospective study of why they failed as a practical agent, in most cases they are no longer a study of a bioherbicide. Therefore, as stated by Fravel (2005), there should be greater communication between researchers and industry earlier in the developmental phase. So if researchers should interact with industry, what industry should they target and at what point in the research cycle? As bioherbicides are often viewed as analogous to synthetic herbicides in many respects (Crump et al., 1999), companies producing and marketing herbicides are the obvious type of commercial partner. Lidert (2001) was one of the many who have suggested the commercialization of bioherbicides through agricultural chemical companies. Many of the large agricultural chemical companies concentrate on the production of synthetic herbicides for a small number of crops on a global basis and therefore are not interested in niche markets for bioherbicides. Combined with this is their need to centralize production and their relative inexperience with pesticides with a live active ingredient. These factors often make

them unsuitable commercialization partners. In many cases, a combination of small to medium sized enterprises (SMEs) with a mixture of production and marketing expertise might present a better business model. With this are the perils of under capitalization of research into these bioherbicides by SMEs. Bioherbicides are inherently difficult to commercialize due to the combination of technologies required to produce them and the need for a mature market into which to introduce the product. At this stage of development researchers should not underestimate the time and money required to bring a product to market in an effort to engage a commercialization partner. Often in an effort to protect their intellectual property and to attract investors, researchers will patent their bioherbicides or part of the technology. This affords protection or privilege to develop the technology for a period of 20 years in most cases (Montesinos, 2003) or 14 years for ‘‘design” patents. Patents are also a form of publication, and so by lodging a patent, the applicant must disclose much of their research data. For a patent to be successful, its lodgement must predate any form of publication of the data. Costs of preparing and lodging patents vary, but escalate based on the number of countries in which the applications are made. Furthermore, as the patent of microorganisms is regulated by the Budapest Treaty7 (28th April, 1977), a pure culture must be lodged with, and maintained by, an officially recognized microbial culture collection (Montesinos, 2003). This is an additional cost to the applicant. In many cases the patenting of bioherbicidal agents is not warranted as the intellectual property resides in the organisms themselves and the fermentation and formulation is often the substance of trade secrets. Unless the market was very lucrative, the effort involved in reverse engineering a bioherbicide would be prohibitive. One of the greatest costs in the commercialization of bioherbicides is in registration. The registration of pesticides is controlled by different organizations within different countries (e.g. the Environmental Protection Agency in the USA, the Australian Pesticides and Veterinary Medicines Authority and the Pest Management and Regulatory Agency in Canada). In general there has been a movement to streamline the registration of biopesticides and a harmonization of requirements between countries (Neale, 2000; OECD, 2003). Assessments have to be made on the threats to human health including toxicological aspects, non-target effects and spread of the proposed biopesticide (Bourdot et al., 2006a; de Jong et al., 2002; Hoagland et al., 2007b). When considering the commercialization of a bioherbicide, companies need to take all of these costs into account.

6. Conclusion A review such as this makes it immediately obvious the breadth and depth of information and techniques which must be mastered in the development of bioherbicides. It is also apparent that much of the research is specific to particular host/pathogen systems and that there are few studies which are truly integrative and allow wider conclusions to be drawn. In the early stages of discovery and development of biopesticides there are innumerable opportunities for research into the basic biology of plant–pathogen interactions, under the guise of development of new sustainable bioherbicides. This partially explains the plethora of articles on ‘‘potential bioherbicides”. Perhaps until an agent is commercialized it should be referred to as a biological control agent and the term bioherbicide or mycoherbicide should be reserved for the formulated, marketed and commercialized product. To move a research 7

http://www.wipo.int/treaties/en/registration/budapest/trtdocs_wo002.html.

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project from the potential to the realization of potential requires large groups with a variety of expertises. Coordination of research in biological control has been approached from a number of angles. In the USA, the Cooperative State Research Service initiated a series of cooperative projects culminating in the S-268 project (Quimby et al., 2002). The aim of this project was ‘‘to develop and utilize some bioherbicide agents that have been previously demonstrated to be effective in preliminary field trials”8 and involved scientists from the USA, Canada, Australia, Great Britain, Israel and Japan (Quimby et al., 2002). Although this has provided an important forum for the exchange of ideas, it has led to few commercial products.(Quimby et al., 2002). Additionally, informal networks such as the International Bioherbicide Group9 which circulate a biannual newsletter and organizes international workshops on bioherbicides will continue to have a great impact on the sharing of knowledge and networks among researchers with an interest in biocontrol of weeds. There are few locations worldwide which have this critical mass and expertise to research, develop and deliver biopesticides. Canada has moved significantly in this direction with the formation of a ‘‘biopesticide innovation chain” (Bailey et al., 2010) and in New Zealand there is the Bio-Protection Centre10. Centralization like this provides a critical mass of researchers and infrastructure which can be used across the full range of biopesticides. These types of groups are often associated with one or more SMEs who provide input into commercialization at early stages of research. This type of model allows the checking at each stage of development of the commercial potential of the product and so a gauge of the real potential for the research to develop a bioherbicide. Greater interaction is also required with industries outside of the traditional pesticide industries such as the food and pharmaceutical industries as advocated by Hynes and Boyetchko (2006). It should be noted that all five of the recently registered bioherbicides in the U.S. and Canada (Woad Warrior; Sarritor, Chontrol, MycoTech, and Smolder), were developed and registered by small-business enterprises or a subsidiary of enterprises with no prior record in pesticide development. Rather than purely technical challenges suggested by Auld and Morin (1995), the future of bioherbicides rests with greater collaboration between a wide variety of scientific disciplines and the early and continued input of industry in the process from selecting the correct agent, target weed through to the selection of the business model. This interaction, however, must be backed by real funds in terms of infrastructure and personnel by public institutions. This is even more imperative in the current global financial situation as commercial companies become more risk averse. References Abu-Dieyeh, M.H., Watson, A.K., 2007. Efficacy of Sclerotinia minor for dandelion control: effect of dandelion accession, age and grass competition. Weed Research 47, 63–72. Agbessi, S., Beausejour, J., Dery, C., Beaulieu, C., 2003. Antagonistic properties of two recombinant strains of Streptomyces melanosporofaciens obtained by intraspecific protoplast fusion. Applied Microbiology and Biotechnology 62, 233–238. Agrios, G.N., 2005. Plant Pathology. Elsevier Inc., Burlington, MA. Aiuchi, D., Baba, Y., Inami, K., Shinya, R., Tani, M., Kuramochi, K., Horie, S., Koike, M., 2007. Screening of Verticillium lecanii (Lecanicillium spp.) hybrid strains based on evaluation of pathogenicity against cotton aphid and greenhouse whitefly, and viability on the leaf surface. Japanese Journal of Applied Entomology and Zoology 51, 205–212. Aly, R., 2007. Conventional and biotechnological approaches for control of parasitic weeds. In Vitro Cellular & Developmental Biology-Plant 43, 304–317. Amsellem, Z., Cohen, B.A., Gressel, J., 2002. Engineering hypervirulence in a mycoherbicidal fungus for efficient weed control. Nature Biotechnology 20, 1035–1039. 8 9 10

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