Discovery of a new strain of Heterorhabditis bacteriophora and use of the inbred line approach to optimize its virulence and cold tolerance

Transcription

1 Discovery of a new strain of Heterorhabditis bacteriophora and use of the inbred line approach to optimize its virulence and cold tolerance by Shahram Sharifi-Far A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Environmental Sciences Guelph, Ontario, Canada Shahram Sharifi-Far, April, 2016

2 ABSTRACT Discovery of a new strain of Heterorhabditis bacteriophora and use of the inbred line approach to optimize its virulence and cold tolerance Shahram Sharifi-Far University of Guelph, 2016 Advisor: Rebecca H. Hallett Co-Advisor: David I. Shapiro-Ilan Serial culturing which leads to trait deterioration is a significant concern in the entomopathogenic nematode production industry for both in vivo and in vitro methods. Prior research indicates that generation of homozygous inbred lines can deter trait loss within a nematode population. I hypothesized that the inbred line approach can be combined with discovery (isolation of new genetic material) to create stable nematode strains with superior biocontrol potential. My objective was to test this hypothesis, in relation to developing superior virulence in colder temperatures. A putative cold-tolerant strain of Heterorhabditis bacteriophora was isolated from a local soil sample in southern Ontario. Ten homozygous inbred lines were generated and screened for biocontrol characteristics, including reproductive capacity, storage stability and virulence in a series of different temperatures. Superiority of the inbred lines compared to their wild-type parents, whether sub-cultured or non-cultured, as well as two commercial strains, was observed. Inbred line 415 exhibited very poor virulence, storage stability, reproductive capacity in small-scale and serial culturing trials, whereas inbred line 324 had the best storage stability, high virulence and reproductive capacity among all passages. Hence, this superior line inbred line 324 was selected for commercial use. Furthermore, the hypothesis that discovery can be combined

3 with the inbred line approach to develop stable and superior nematodes for biocontrol was supported.

4 Acknowledgements I would like to take this opportunity to express my heartfelt gratitude to my coadvisors Drs. Rebecca Hallett and David I. Shapiro-Ilan for their consistent support and continuous encouragement throughout my thesis research. Rebecca was the person that opened the door for me to build a better future for my profession. She broadened my confidence extensively and encouraged me throughout the long process. I am forever grateful for her leadership and motivation. David was there every step of the way and helped me overcome every obstacle that I faced. His mentorship and vast knowledge on this study was a great asset to me and this research would have been impossible without his supervision. I would like to express my profound gratitude to my third advisory committee member, Dr. Michael Brownbridge, whose guidance made my research more efficient. I was very lucky that he supported me in this project and offered me all the assistance and encouragement that I needed. The things he did for me were priceless and I am forever grateful. Thank you to Dr. Stephen Bowley who helped me with the statistical analysis and using the SAS program. His fast replies were a lifesaver. I am forever thankful to Dr. Hossein Moosavinia, my teacher in Ahwaz University of Iran, who inspired and reinforced me, making me believe that I could reach my dreams and continue my studies even after immigration. A special thank you goes out to Susan Cavey and John Robertson for believing in me and cheering me on throughout this study. I am indebted to Kim Bauer, my colleague, for all her hard work and continuous assistance in the study. Also my friend Braden Evans, who strongly supported me in the laboratory tasks at University of Guelph. iv

5 This would not have been possible without the support of my family, especially my beautiful wife, Mehri Keshavarz, who has put up with me and has been my rock throughout a long journey and my life in general, my lovely daughter Minoo and my clever son Maney. I would like to thank my nephew, Amin Soleimani, who was available anytime I needed him, regardless of the five hour time difference. I would like to dedicate this study to the late Sandra Mitchell who started this journey with me five years ago, but has sadly left the world too soon. She and her husband, Mr. David Mitchell, encouraged me to broaden my horizons and allowed me to study and continue my career at the same time. Finally, I am ceaselessly appreciative to my parents for always believing in me and pushing me to chase my dreams. This achievement would not have been possible, if it weren t for the constant aid of everyone who worked or currently works at Natural Insect Control and walked this long journey with me. v

6 Table of Contents ABSTRACT... ii Acknowledgements.iv Table of Contents... vi List of Tables... viii List of Figures... ix Table of Acronyms... xiii CHAPTER ONE: Literature Review 1.1 Introduction Entomopathogenic nematodes Life cycle Photorhabdus and Xenorhabdus The relationship between entomopathogenic nematodes and their symbiotic bacteria Phase variation Mass production of entomopathogenic nematodes In vivo production In vitro production Genetic improvement Selective breeding Hybridization Mutagenesis Discovery of new strains or species Methods of genetic engineering Trait deterioration Trait stabilization Cryopreservation to maintain beneficial traits Research objectives CHAPTER TWO: Creating homozygous inbred lines from a Canadian strain of Heterorhabditis bacteriophora 2.1 Abstract Introduction Materials and methods Local strain extraction Media preparation for bacterial culture Inbred line development Duration of the inbred lines life cycle Efficacy of inbred lines Results Discussion vi

7 CHAPTER THREE: The impact of serial culturing on fitness and production capacity of entomopathogenic nematodes: inbred lines vs. wild-type populations 3.1 Abstract Introduction Materials and methods Small-scale production trial Large-scale production trial Storage stability trial Serial culturing trial Statistical analyses Results Small-scale trial Large-scale trial.... Error! Bookmark not defined Storage stability trial Serial culturing trial Discussion CHAPTER FOUR: Efficacy of homozygous inbred lines of Heterorhabditis bacteriophora against the greater wax moth, Galleria mellonella, and cabbage maggot, Delia radicum, under relatively cold conditions 4.1 Abstract Introduction Materials and methods Cool temperature assessment trial against Galleria mellonella Efficacy of inbred lines against Delia radicum under cool conditions Statistical analyses Results Cool temperature assessment trial against Galleria mellonella Efficacy of inbred lines against Delia radicum under cool conditions Root damage assessment results Discussion CHAPTER FIVE: General Discussion and Recommendations..99 References vii

8 List of Tables Table 1.1. Insect pests targeted for control using entomopathogenic nematodes. This table includes major target pests and the nematodes used to control them based on industry recommendations and refereed scientific articles indicating high levels of efficacy; the table is not meant to be an exhaustive list (Shapiro-Ilan and Gaugler, 2002; Grewal et al., 2005; Georgis et al., 2006; Lacey and Georgis, 2012; Lacey and Shapiro-Ilan, 2008)...9 viii

9 List of Figures Figure 2.1. Inbred line development procedure; two steps including hermaphrodite selection and individual juveniles transfer would be repeated for seven times to create homozygous population 30 Figure 2.2. Haemolymph collection procedures. (A) The inoculated and surface-sterilized Galleria mellonella larva was held at its dorsal side with curved forceps, with gentle pressure and angling the head downwards. (B) After cutting of the middle leg, haemolymph squirts out. (C) Haemolymph is collected by a sterile inoculation loop. (D) The haemolymph is streaked on a nutrient agar plate in a four quadrant pattern. Photographs: S. Sharifi-Far...32 Figure 2.3. (A) Four quadrant streak plating of Photorhabdus luminescens on nutrient agar with a single colony of the first phase of symbiotic bacteria visible in the fourth streak zone. (B) Inoculated nutrient agar plates from single colonies (left) and their specific corresponding T7 & TTC media plates (right) (C) Dye adsorption in T7 & TTC media plate confirms that P. luminescens is in phase I. (D) Development of P. luminescens in the nutrient agar plate after one hundred and twenty hours at 25 o C. Photographs: S. Sharifi-Far..34 Figure 2.4. (A) Comparison of Photorhabdus luminescens development on nutrient agar (left) and lipid agar (right). (B) Heterorhabditis bacteriophora hermaphrodites in lipid agar plates at 40 times magnification. (C) Lipid agar plates with hermaphrodites, five days after introduction of surface sterilized infective juveniles. (D) A centrifuge tube with washed hermaphrodites before egg extraction process. Photographs: S. Sharifi-Far...37 Figure 2.5. Mean duration of life cycle in days (± SE) of a local strain of Heterorhabditis bacteriophora in lipid agar media at room temperature (22 o C). The appearance of hermaphrodite in two consecutive generations were considered as the life cycle of the inbred lines. Columns with the same letter are not significantly different (P 0.05) Figure 2.6. Mean percentage (± SE) efficacy of homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to non-cultured and sub-cultured populations of their wild-type parents, against the larval stage of Galleria mellonella at 22 o C. The subcultured population 15 passages of serial culturing through G. mellonella, while the noncultured population was the original population collected after soil extraction. Columns with the same letter are not significantly different (P 0.05). 43 Figure 3.1. Mean (± SE) reproductive capacity per cadaver of the larval stage of Galleria mellonella in the small-scale production trial, conducted at 22 o C, with ten homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to non-cultured and sub-cultured populations of their wild-type parents and two commercial strains of the same species from an American (Com 1) and European (Com 2) producer. The sub-cultured population went through 15 passages of serial culturing through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Columns with the same letter are not significantly different (P 0.05) ix

10 Figure 3.2. Mean (± SE) reproductive capacity per cadaver of the larval stage of Galleria mellonella in the large-scale production trial, conducted at 25 o C, with ten homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to non-cultured and subcultured populations of their wild-type parents and two commercial strains of the same species from an American (Com 1) and European (Com 2) producer. The sub-cultured population went through 15 passages of serial culturing through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Columns with the same letter are not significantly different (P 0.05) Figure 3.3. Mean (± SE) % mortality of homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to non-cultured and sub-cultured populations of their wild-type parents and two commercial strains of the same species from an American (Com 1) and European (Com 2) producer, which were aerated and stored at 13 o C for A) 50, B) 65, and C) 80 days. Columns within a figure with the same letter are not significantly different (P 0.05) Figure 3.4. Assessment of the effect of serial culturing on the virulence (mean ± SE percent efficacy) of homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to non-cultured and sub-cultured populations of their wild-type parents, against the larval stage of Galleria mellonella, after the A) first, B) fifth, C) tenth, and D) fifteenth passage through G. mellonella at 22 o C. Columns within a figure with the same letter are not significantly different (P 0.05)...62 Figure 3.5. Assessment of the effect of serial culturing on the virulence (mean ± SE percent efficacy) of individual homozygous inbred lines from a local strain of Heterorhabditis bacteriophora over the culture intervals compared to non-cultured and sub-cultured populations of their wild-type parents, against the larval stage of Galleria mellonella at 22 o C after the first, fifth, tenth and fifteenth passages through G. mellonella (P 0.05) Figure 3.6. Mean reproductive capacity (± SE) of homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to non-cultured, sub-cultured populations of their wild-type parents, per cadaver (x 1000), influenced by serial culturing after first (A), fifth (B), tenth (C) and fifteenth (D) passage, through the larval stage of Galleria mellonella at 22 o C (P 0.05).66 Figure 3.7. Mean reproductive capacity (± SE) of each individual homozygous inbred line from a local strain of Heterorhabditis bacteriophora and their non-cultured, sub-cultured populations of the wild-type parents, per larval stage of the Galleria mellonella cadaver (x 1000), influenced by serial culturing after first, fifth, tenth and fifteenth cultured intervals through the larval stage of G. mellonella at 22 o C (P 0.05)...69 Figure 4.1. The average monthly temperature ( o C) in Toronto, Ontario, based on climate data gathered from 1995 to 2014 ( 2015). The three generations of cabbage maggot, Delia radicum, that occur in southern Ontario are represented by the bars above the graph 77 x

11 Figure 4.2. Rearing container for Delia radicum maggots on rutabaga media (A) Top view of D. radicum rearing cage, containing water containers with rolled filter paper wicks to provide the desired moisture. (B) Oviposition device, consisting of organic rutabaga pieces and activated charcoal placed on a petri dish and covered with inverted clay pots. (C) Deliaradicum eggs were collected on a fine screen. (D) Delia radicum maggots were fed on a piece of an organic rutabaga. Photographs: S. Sharifi-Far Figure 4.3. (A) Ten first instar Delia radicum ready to be introduced to an experimental pot. (B) Artificial infestation of cabbage plant by introducing cabbage maggot, D. radicum, to the prepared trench and recovering the soil. (C) Placing the experimental pots in the incubator at 16 o C with 14:10 hours day/night photoperiodism. (D) Broccoli root system investigated for maggot damage classification. Photographs: S. Sharifi-Far.83 Figure 4.4. Mean percentage (± SE) efficacy of homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to the non-cultured and sub-cultured populations of their wild-type parents, against the larval stage of Galleria mellonella at 8 o C. The subcultured population underwent 15 passages of serial culturing through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Columns with the same letter are not significantly different (P 0.05). There is no difference between treatments at this temperature. 86 Figure 4.5. Mean percentage (± SE) efficacy of homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to the non-cultured and sub-cultured populations of their wild-type parents, against the larval stage of Galleria mellonella at 12 o C. The subcultured population underwent 15 passages of serial culturing through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Columns with the same letter are not significantly different (P 0.05). Columns with the same letter are not significantly different (P 0.05). There is no difference between treatments at this temperature...87 Figure 4.6. Mean percentage (± SE) efficacy of homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to the non-cultured and sub-cultured populations of their wild-type parents, against the larval stage of Galleria mellonella at 16 o C. The subcultured population underwent 15 passages of serial culturing through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Columns with the same letter are not significantly different (P 0.05)...88 Figure 4.7. Mean percent (± SE) efficacy of homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to the non-cultured and sub-cultured populations of their wild-type parents, against the larval stage of Galleria mellonella at 20 o C. The subcultured population underwent 15 passages of serial culturing through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Columns with the same letter are not significantly different (P 0.05)..89 xi

12 Figure 4.8. Mean percent (± SE) efficacy of homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to the non-cultured and sub-cultured populations of their wild-type parents, against the larval stage of Galleria mellonella at 24 o C. The subcultured population underwent 15 passages of serial culturing through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Columns with the same letter are not significantly different (P 0.05).90 Figure 4.9. Mean percent (± SE) mortality of against the larval stage of cabbage maggot, Delia radicum, homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to the non-cultured and sub-cultured populations of their wild-type parents, and to two commercial strains at 16 o C. Each treatment was applied at two rates low (L, 4000 IJs/pot) and high (H, 40,000 IJs/pot). The control treatment only received 50 ml. of distilled water. Columns with the same letter are not significantly different (P 0.05) Figure Mean of qualitative point (± SE) of root system development after five weeks. The best condition was valued as five and the worst damaged roots received one point. The cabbage maggot, Delia radicum, damage control in 15 cm diameter pot throughout homozygous inbred lines from a local strain of Heterorhabditis bacteriophora compared to the non-cultured and sub-cultured populations of their wild-type parents, also two commercial strains at 16 o C were examined. Each treatment was applied at two rates, low (L, 4000 IJs/pot) and high (H, 40,000 IJs/pot). The control treatment only received 50 ml. of distilled water. Columns with the same letter are not significantly different (P 0.05) xii

13 Table of Acronyms cfu colony-forming units EPN entomopathogenic nematode IJ infective juvenile stage J1 first juvenile stage J2 second juvenile stage J3 third juvenile stage J4 forth juvenile stage PCR polymerase chain reaction RH relative humidity SE standard error T7 tergitol 7 agar TTC 2, 3, 5-triphenyl tetrazolium chloride UV ultraviolet radiation xiii

14 CHAPTER ONE Literature Review 1.1 Introduction Biological control utilizes beneficial organisms to achieve the suppression of pest populations below economically damaging levels, and is considered an environmentally safe and effective component of integrated pest management (Higley and Pedigo, 1996). To be successful, a biocontrol agent must possess several important traits, such as, virulence to the target pest and environmental competence (Hopper et al., 1993). Biocontrol success may be enhanced through the selection of individuals demonstrating desirable traits. Furthermore, once an efficient biocontrol agent is developed for commercial use, maintenance of beneficial traits is also essential to ensure consistency of performance (Glazer et al., 1991; Hopper et al., 1993). When a biological control agent is repeatedly produced in culture, certain important biological characteristics can be lost or diminish due to genetic or non-genetic factors (Bai et al., 2005; Adhikari et al., 2009). Inbreeding, drift, or inadvertent selection has a genetic basis (Roush, 1990; Hopper et al., 1993), while disease and malnutrition are non-genetic factors that can potentially cause trait variation (Hopper et al., 1993). Ultimately, all of these factors can negatively affect performance, which leads to loss of confidence in a commercial product with subsequent impact on sales and revenue. Trait deterioration has been reported extensively in a range of different arthropods. Female fecundity in the bulb mite, Rhizoglyphus robini (Astigmata: Acaridae), was reduced in experimental lines after 11 generations, when males competed for females in a subdivided 1

15 population compared with the control treatment in which several hundred mites are kept in a container, naturally. Inbreeding depression throughout the male population may have played a role in this reduction (Radwan et al., 2004). Reduced fecundity, longevity and size have been observed in Drosophila melanogaster (Diptera: Drosophilidae), with high levels of inbreeding (Tantawy & Reeve, 1956). Maternal effects (i.e. non-genetic factors, such as nutrition and disease) must also be considered in trait deterioration studies (Hopper et al., 1993). Trait enhancement and deterioration have been widely reported in entomopathogenic nematodes (EPNs) (Shapiro-Ilan et al., 2012). Therefore, EPNs are considered good model organisms for studying trait improvement and stabilization. One technique used to achieve trait stability in living creatures is the creation of homozygous inbred lines. Using this technique, some of the lines produced have a lower level of unfavourable traits than their parents (Roush, 1990). This method also has the ability to defend against the loss of beneficial traits caused by a variety of factors such as inbreeding depression and inadvertent selection during the serial culturing of an organism (Hopper et al., 1993). The objective of this research project was to use the inbred line procedure as a method for genetic stabilization and improvement of a Canadian strain of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), with a specific goal of enhancing the expression of beneficial characteristics, including low temperature efficacy and virulence. 1.2 Entomopathogenic nematodes Entomopathogenic nematodes are classified in the phylum Nematoda, the roundworms. The term entomopathogenic is derived from the Greek word entomon 2

16 defined as insect and pathogenic referring to illness (Gaugler, 2006). Entomopathogenic nematodes enter an insect host s body and, with the assistance of symbiotic bacteria, develop, reproduce and cause the death of the host (Bedding and Molyneux, 1982; Poinar, 1990; Akhurst and Dunphy, 1993). There are two main genera of EPNs, Steinernema and Heterorhabditis (Poinar, 1975), which carry two different symbiotic bacteria in the anterior part of their intestine: Xenorhabdus spp. and Photorhabdus spp., respectively (Akhurst and Dunphy, 1993). After host penetration, the symbiotic bacteria are injected into the host haemocoel, where they quickly reproduce and typically kill the host by septicaemia or toxaemia within 48 hours (Chaston and Goodrich-Blair, 2010). Steinernema and Heterorhabditis will be used here to discuss elements of the life cycle and bacterial associations of EPNs Life cycle Similar to the majority of nematodes, both Steinernema spp. and Heterorhabditis spp. have a simple life cycle that includes the egg, four juvenile (J1 to J4) and adult stages (Woodring and Kaya, 1988). However, the infective juvenile (IJ) stage is unique to EPNs (Poinar, 1990). The IJ is a specialized J3 that has the sheath of the J2 stage still covering it. Infective juveniles can enter the host insect s body via the mouth, anus, and spiracles, or by scraping the cuticle and penetrating directly into the host cavity (Bedding and Molyneux, 1982; Poinar, 1990; Akhurst and Dunphy, 1993; Peters and Ehlers, 1994). Amphimictic and automictic reproduction. After penetrating the host body Steinernema IJs moult through the J3 and J4 stages and develop into adult males or females, i.e. amphimictic reproduction (Poinar, 1990). However, in Heterorhabditis hermaphrodite 3

17 adults are formed (Woodring and Kaya, 1988; Strauch et al., 1994). In this genus, the subsequent generations may be a combination of amphimictic and self-fertilizing (i.e. automictic) forms (Strauch et al., 1994; Koltai et al., 1995) that develop into IJs (Johnigk and Ehlers, 1999). Reproduction continues for one to three generations inside the host body (Poinar, 1990), depending on host size and species (Jackson, 1985). The next generation of juveniles is created by the adults when there are sufficient nutrients remaining from the host tissues to sustain them (Strauch et al., 1994). In contrast, if the host resources are consumed and conditions become unfavourable for further reproduction, IJs will be produced (Adams and Nguyen, 2002), which leave the insect cadaver to look for new hosts (Poinar, 1990; Glazer et al., 1991; Strauch and Ehlers, 2000). Infective juveniles. The IJs are a free-living stage, and are the only form of EPNs that are commercially available for biological control purposes (Shapiro-Ilan and Gaugler, 2002). In the transition from the second juvenile stage (J2) to the IJ stage, no shedding of the cuticle occurs, resulting in a double layer of cuticle, which may help IJs tolerate less than optimal environmental conditions (Woodring and Kaya, 1988). The IJ stage is analogous to the dauer form of Caenorhabditis elegans (Rhabditida: Rhabditidae; Poinar, 1975), which lives in manure and soil as a free-living bacterial feeding nematode. Entomopathogenic nematodes and C. elegans belong to the same order; therefore genome studies and genetic tools that have been developed for C. elegans are important assets for EPN research (Burnell, 2002; Ciche and Ensign, 2003). Endotokia matricida. As mentioned above, when the food supply is limiting, IJs are created in an infected host. Reduction of nutrients triggers the termination of oviposition and stimulates the occurrence of endotokia matricida. Normally, a female EPN produces eggs 4

18 that are deposited in the host body where they hatch. However in the process of endotokia matricida, eggs hatch within the EPN uterus as J1s that develop into the IJ form (Johnigk and Ehlers, 1999 and Baliadi et al., 2001). When IJs are fully developed, they burst through the wall of the uterus, thus killing their mother. The availability of nutrients postpones the occurrence of endotokia matricida and stimulates continued oviposition (Johnigk and Ehlers, 1999). The intestinal tissues of the IJs are very similar to those of adults and contain symbiotic bacteria cells and stored nutrients (Johnigk and Ehlers, 1999). Infective juveniles also contain large quantities of lipids that are important energy resources and are critical to their success as the free-living host-seeking stage (Johnigk and Ehlers, 1999). This specific form of IJs, which has evolved through endotokia matricida, occurs in both EPN genera (Ehlers, 2001). Endotokia matricida has been studied extensively in three EPN species: S. glaseri (Rhabditida: Steinernematidae), S. carpocapsae (Rhabditida: Steinernematidae) and H. bacteriophora. The majority of IJs in S. carpocapsae (64%) and H. bacteriophora (81%) are produced by endotokia matricida compared to normal growth stages in which females oviposit outside of the body chamber (Baliadi et al. 2001). In S. glaseri, a total of 28% of IJs are developed by endotokia matricida. The third adult generation contributes a significant proportion of the IJ population by endotokia matricida, i.e. 24%, 51% and 63% for S. glaseri, S. carpocapsae and H. bacteriophora, respectively (Baliadi et al. 2001). There is a correlation between the degree of endotokia matricida occurring in the culture of EPNs and IJ production levels (Ehlers, 2001). 5

19 Recovery. Once IJs penetrate a host, they develop into J3s, followed by the J4 and adult stages. The transition from the IJ to J3 stage is called recovery. A critical trigger for recovery is exposure of IJs to the haemolymph of their host, which signals the presence of food (Strauch and Ehlers, 1998). Symbiotic bacteria are discharged by EPNs at this time and, once the host is dead, they multiply and subsequently with digestion of the haemolymph and internal tissues of the host, they provide the primary source of nutrients for the EPNs (Hirao and Ehlers, 2009). The proportion of IJs entering recovery in vivo is often 100% in heterorhabditids (Strauch and Ehlers, 1998). The recovery rate can fluctuate between 18 and 90 % (Strauch and Ehlers, 1998), for in vitro liquid culture depending on the nutrients available, aeration, carbon dioxide concentration, temperature and lipid quantity (Ehlers et al., 2000) Photorhabdus and Xenorhabdus Bacterial colonies in both Steinernema spp. and Heterorhabditis spp. are located between the pharynx and the tail of the nematodes. Xenorhabdus cells are concentrated in a vesicle, whereas Photorhabdus cells reside in a large portion of the lumen of the nematode intestine (Goodrich-Blair and Clarke, 2007). Heterorhabditis bacteriophora IJs discharge their symbiotic bacteria, P. luminescens (Enterobacteriales: Enterobacteriaceae) after incubation in haemolymph for 30 minutes (Ciche and Ensign, 2003). After 30 min, the bacteria are discharged constantly, at a steady rate for more than 5 h (Ciche and Ensign, 2003). These bacteria have both mutualistic (inside the IJ) and parasitic (inside the host) associations during their life cycle. Within the pathogenic association, the diseased host becomes a food supply for both organisms; the 6

20 bacteria assimilate insect organs into a digestible soup for the nematodes (Goodrich-Blair and Clarke, 2007). The approximate number of bacterial cells located within the anterior part of the nematode intestine is colony-forming units (cfu) for Photorhabdus (Ciche and Ensign, 2003), and cfu for Xenorhabdus (Martens et al., 2003). There is no significant difference in location and quantity, of P. luminescens observed between fresh and aged (30 days) IJs. Thus, bacterial reproduction and growth may be restricted in the IJ intestine The relationship between entomopathogenic nematodes and their symbiotic bacteria The relationship between EPNs and their symbiotic bacteria is an example of classic mutualism, where both the EPN and bacterium profit from their relationship (Akhurst, 1982). Xenorhabdus and Photorhabdus bacteria cannot live freely in the soil, excluding P. asymbiotica (Boemare and Akhurst, 2000), and are thus dependent on being protected within the body of the IJ. Residing within the EPN intestine allows the bacteria to be transported into a host insect and protected from antibacterial host compounds (Akhurst and Boemare, 1990). Although, the symbiotic bacteria exhibit tolerance against the host immune system such as haemolymph enzymes that can cause bacterial cell wall collapse upon bacteria infection (Dunn 1986, Forst and Nealson, 1996). The bacteria kill the host rapidly so that the host tissue and bacterial cells become a food supply to sustain the nematode (Akhurst and Boemare, 1990). In addition, the bacteria produce antibiotics that protect the nematodes from other microorganisms (Akhurst, 1982; 7

21 Hu and Webster, 2000). The nematode-bacterium complex acts in concert to attack and kill the larval stages of a wide range of insect pests, such as white grubs, Phyllophaga spp. (Coleoptera: Scarabaeidae) and root weevils e.g. Otiorhynchus sp. (Coleoptera: Curculionidae) (Akhurst and Dunphy, 1993). Examples of pest insects that can be infected by EPNs are listed in Table 1.1 (Shapiro-Ilan et al., 2014). There is a correlation among EPNs and bacterial species (Akhurst & Boemare, 1988); although all EPNs only associate with one species of bacteria, some bacteria can be associated with multiple EPN species (Akhurst and Boemare, 1990). Additionally, at a lower taxonomic level rank than species, which is referred to as a strain, each nematode requires a specific strain of bacterium to develop. The specificity between nematode species and symbiotic bacterium strain or sub-species has been studied; association between nematodes and undesired bacteria can compromise efficacy, survival (Chapuis et al., 2009), or impact maintenance of symbiosis (Murfin et al., 2015) Phase variation Both Photorhabdus and Xenorhabdus have two pleomorphic forms, which have different cultural, physiological and morphological characteristics (Akhurst, 1980; Ehlers, 2001; Owuama, 2001). These morphs are referred to as phase I and phase II or primary and secondary forms, respectively. Phase I bacteria are associated with IJs in natural conditions and develop when in vivo culture conditions are optimized (Ehlers, 2001). Phase II bacteria occur when bacteria are propagated in suboptimal conditions, e.g. high temperatures and insufficient oxygen (Krasomil-Osterfeld, 1995; Ehlers, 2001). 8

22 Table 1.1. Insect pests targeted for control using entomopathogenic nematodes. This table includes some of the major target pests and nematodes used to control them based on industry recommendations and refereed scientific articles where high levels of efficacy were obtained (Shapiro-Ilan and Gaugler, 2002; Grewal et al., 2005; Georgis et al., 2006; Lacey and Georgis, 2012). Also see Grewal et al., 2005; Georgis et al., 2006; Lacey and Shapiro-Ilan, Common Name Scientific Name Nematode(s) * Artichoke plume moth Platyptilia carduidactyla (Riley) Sf, Sc Banana moth Opogona sachari (Bojer) Hb, Sc Banana root borer Cosmopolites sordidus (Gemar) Sc, Sf, Sg Billbug Sphenophorus spp. Sc Black cutworm Agrotis ipsilon (Hufnagel) Sc, Hb Black vine weevil Otiorhynchus sulcatus (F.) Hb, Hmg, Sk Blue green weevils Pachneus spp. Sr, Hb Borers Synanthedon spp. (Lepidoptera: Sesiidae) Sc, Hb, Sf Cabbage maggot Delia radicum (L.) Sf Cat flea Ctenocephalides felis (Bouché) Sc, Hb Chinch bug Blissus leucopterus (Say) Sc Codling moth Cydia pomonella (L.) Sf, Sc Corn rootworm Diabrotica spp. Hb, Sf Cranberry girdler Chrysoteuchia topiaria (Zeller) Sc Diamondback moth Plutella xylostella (L.) Sc Diaprepes root weevil Diaprepes abbreviatus (L.) Sr, Hb, Hi Fungus gnats Diptera: Sciaridae Sf, Hb Large pine weevil Hylobius abietis (L.) Hd, Sc, Sf Leafminer Liriomyza spp. (Diptera: Agromyzidae) Sf, Sc Mole crickets Scapteriscus spp. (Orthoptera: Gryllotalpidae) Ss, Sr, Sc Navel orangeworm Amyelois transitella (Walker) Sc Pecan weevil Curculio caryae (Horn) Sc Plum curculio Conotrachelus nenuphar (Herbst) Sr Small hive beetle Aethina tumida (Murray) Sr, Hi Strawberry root weevil Otiorhynchus ovatus (L.) Hb, Hmr Sweetpotato weevil Cylas formicarius (F.) Hb, Hi Western flower thrips Frankliniella occidentalis (Pergande) Sf White grubs Coleoptera: Scarabaeidae Hb, Hmg, Sg * Hb = Heterorhabditis bacteriophora, Hd = H. downesi (Stock, Burnell and Griffin), Hi = H. indica, Hmr = H. marelatus, Hmg = H. megidis, Sc = Steinernema carpocapsae, Sf = S. feltiae, Sg = S. glaseri, Sk = S. kraussei, Sr = S. riobrave, Ss = S. scapterisci. 9

23 The lipase levels, protease activity and quantity of extracted antibiotics in phase I Photorhabdus are significantly higher than in phase II (Boemare and Akhurst, 1988; Forst and Nealson, 1996). The deficiency of these substances can affect the developmental rate and reproductive capacity of EPNs (Han and Ehlers, 2001). An exception to this is found in Xenorhabdus nematophila (Enterobacteriales: Enterobacteriaceae), where the lipase activities in phase II cells are higher than in phase I cells (Boemare and Akhurst 1988). When both phases of bacteria are available, different strains of IJs accept both, but show greater preference for the primary phase over the secondary phase (Gerritsen and Smits, 1997). Further understanding of phase variation should lead to a greater appreciation of its crucial effects on the nematode-bacteria complex; e.g. phase I is the only form that can produce antibiotics, which is a characteristic that can dramatically affect nematode virulence (Boemare and Akhurst, 1988; Forst and Nealson, 1996). 1.3 Mass production of entomopathogenic nematodes Mass production of EPNs can be categorized into two main methods: in vivo, in which nematodes are reared in an insect host, and in vitro, in which reproduction occurs in artificial media (Shapiro-Ilan and Gaugler, 2002). When selecting an appropriate method, a number of factors must be considered including technical capability, labour, EPN quantity requirements and species preference for the production method (Woodring and Kaya, 1988) In vivo production In vivo culture is crucial to many scientific and industrial endeavours, even though in vitro production has the benefit of economy of scale. The in vivo production of EPNs is a 10

24 fairly straight-forward process (Friedman, 1990). A wide variety of different methods have been evaluated for in vivo culture of EPNs (Shapiro-Ilan and Gaugler 2002). A common process is based on using White traps (White, 1927), which provide a suitable environment for EPNs after multiplying inside the host cadaver to leave their host cadaver after a certain time (Shapiro-Ilan et al., 2012). The 7 th instar of the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae), is the most commonly used insect host for EPN culture. Galleria mellonella has a high level of susceptibility to a large variety of nematodes; it is also widely accessible, easy to maintain and provides high nematode yields (Woodring and Kaya, 1988; Shapiro-Ilan and Gaugler, 2002). An alternative host for in vivo production is the mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae) (Grewal, et al., 1999). Laboratory scale in vivo production has been described by Woodring and Kaya (1988) and can be used at a commercial level. The key stages of large-scale in vivo production are: inoculation, harvest, amalgamation and sanitization (Shapiro-Ilan and Gaugler, 2002) In vitro production The process of in vitro culturing depends on bringing together the nematodes, in a nutritional medium, with a pure culture of their symbiotic bacteria (Shapiro-Ilan et al., 2012). In this procedure, the symbiotic bacteria are first introduced followed by the nematodes (Buecher and Popiel, 1989; Surrey and Davies, 1996; Strauch and Ehlers, 2000). In vitro production can be divided into solid and liquid culture methods (Shapiro-Ilan et al., 2012). Solid culture. Using different media such as animal organs, dog food, etc. (Hara et al., 1981, Shapiro-Ilan and Gaugler, 2002). Wouts (1981) was one of the first scientists to use 11

25 polyurethane foam for solid culture. He prepared a medium that included nutrient broth, yeast extract, soy flour, corn oil and water. With Robin Bedding s consultation, a thick substrate was soaked into 9 gm of moist foam slices. This procedure yielded up to 10 million IJs/250 ml flask in 30 days (Wouts, 1981). This innovation brought about significant advances in solid-substrate culture methods (Bedding, 1981), such as the mixing of a liquid medium with a crumbled polyether or polyurethane foam (sponge); the mixture is subsequently autoclave-sterilized (Bedding, 1981, 1984). To increase the foam s absorption capacity, Kelgum (Xanthan gum blend), was added to the growth medium (Gaugler and Han, 2002). Subsequently, bacteria are introduced to the media, followed 72 hours later by the nematodes. Within 2 to 5 weeks the nematodes are harvested by placing the foam into sieves and soaking them in water (Bedding 1981 and 1984). The final IJ yield produced through solid culture can vary based on nematode species, duration of process and inoculum volume (Wang and Bedding, 1998). By using bags made from autoclavable polypropylene tubing (instead of Erlenmeyer flasks), and by covering the sponge with a layer of homogenized chicken organs, production of up to 2 billion nematodes per 3 kg bag was achieved (Bedding, 1981, 1984). Liquid culture. The first report of in vitro mass production of EPNs in liquid culture was made by Glaser et al. (1940). However, at that time, the existence of symbiotic bacteria living within the nematode was unknown and was not discovered until many years later (Poinar and Thomas, 1966). Microbial contaminants can have a significant, negative impact on the production of EPNs in liquid culture. To avoid introduction of such contaminants into the culture medium, 12

26 nematodes were initially surface-sterilized prior to their being added to the bacterial (symbiont) culture (Akhurst, 1980; Wouts, 1981). However, as contaminants could remain between the first and second cuticle in spite of the sterilization process, methods were revised to use axenic nematode eggs instead of IJs (Lunau et al., 1993). Infective juveniles are introduced to the medium h after inoculation with the symbiont bacteria, when the bacteria comprise approximately 0.5 to 1 percent of the mass of the culture. Specific procedures for nematode inoculation vary according to media composition and nematode species (Ehlers et al., 2000). Generally, media contain lipid and protein, which may be derived from plant or animal sources, as well as yeast extract (Friedman et al. 1989; Han et al. 1993; Ehlers et al. 1998). In vitro production inside a vessel is a complex process and has to accommodate the interrelated reproduction dynamics of two organisms. Two major issues during production are a change from the first to the second phase of symbiont bacteria, and a postponement or lagging of nematode inoculum development (Ehlers, 2001). The second phase could occur by extending the sub-culture, under an undesired situation, which provides an unsuitable environment for nematodes and impacts their advancement and harvest ratio, which may play an important role in this method s failure (Ehlers et al., 1990; Völgyi et al. 1998; Han and Ehlers, 2001; Floyd et al., 2012). Also, allowing insufficient time for nematode incubation could affect recovery and yield (Johnigk et al., 2004). The recovery of IJs from a culture medium is lower than from a live host (Strauch and Ehlers, 1998). The low rate of recovery might be caused by delayed nematode development which can prolong the culture period, incorrect ph, or other factors, all of which impact productivity (Ehlers et al., 1998; Johnigk et al., 2004). 13

27 An important consideration in the mass production of S. carpocapsae in a bioreactor 1 is achieving maximum fertilization of female nematodes (Neves et al., 2001). Ideally, all female nematodes are placed in one location in the bioreactor to increase the likelihood that they will successfully mate with males. Reducing airflow in the bioreactor results in a decrease in circulation rate and optimizes nematode distribution for mating (Neves et al., 2001). Other essential variables that can affect nematode production in a bioreactor are dissolved oxygen levels, ph, temperature, agitation and airflow (Floyd et al., 2012). In general, production levels for EPNs obtained in a bioreactor can vary according to the components of the medium, nematode species and procedure. For H. bacteriophora, production can range from to IJs/ml (Floyd et al., 2012) and from to IJs/ml for S. carpocapsae (Neves et al., 2001). The highest quantity reported with this method was > IJs/ml for H. indica (Ehlers et al., 2000). 1.4 Genetic improvement Following the discovery of a biocontrol candidate, investigations are often undertaken to determine whether specific characteristics can be altered to improve performance (Gaugler, 1987). Entomopathogenic nematodes have often been the subject of genetic studies due to their short life cycle and relative ease of culture (Burnell, 2002). Improvements in nematode strains and their associated symbionts can promote their commercial potential (Burnell and Dowds, 1996). Genetic improvement of EPNs can be undertaken to increase 1 A standard piece of scientific equipment used for fermentation; typically refers to a kind of fermenter used for larger organisms (nematodes) and cells. 14

28 desirable and decrease undesirable characteristics in order to enhance production characteristics and increase IJ efficacy (Burnell, 2002; Grewal et al., 2005). One of the most effective approaches to improving physiological traits, e.g. environmental tolerance, is selection breeding (Burnell and Dowds, 1996). Heat and cold tolerance, pathogenicity, host specificity, environmental adaptability and control of phase variation are some of the essential objectives for genetic enhancement of Xenorhabdus spp. and Photorhabdus spp. (Burnell and Dowds, 1996). Other qualities that require improvement include virulence and stability in storage/shipping (Kaya, 1985). One example of a genetic improvement program based on a heterogeneous population of S. feltiae resulted in an increase in the EPN s host-seeking ability and tolerance against desiccation (Salame et al., 2010). More intricate studies of the basic biology of EPNs have included genetic aspects that may be leveraged toward improved biocontrol (Dowds, 1994). Genomic sequencing and annotation have been recognized as mechanisms for identifying genes responsible for beneficial biocontrol traits, as well as other critical aspects of the EPN life cycle. Access to the genome sequence of the symbiotic bacteria (e.g. P. luminescens subspecies laumondii strain TT01), can also lead to improvements in nematode performance (Duchaud et al., 2003). For example, details of specific genes involved in the synthesis of antibiotics would be valuable in the selection of more host-competent strains; similarly, knowledge of gene function can lead to a better understanding of the relationship between nematode pathogenesis and their associated symbionts, which could lead to improvements in activity (Duchaud et al., 2003). Research on the genome of H. bacteriophora confirmed a similarity between this species and free-living species (i.e. C. elegans), and plant and human parasitic 15

29 nematodes, including Meloidogyne hapla (Meloidogynidae: Tylenchida) and Brugia malayi (Spirurida: Onchocercidae), respectively (Bai et al., 2013). Using the well-studied organisms such as the above nematodes or Escherichia coli could be a shortcut for a better understanding of biological phenomena e.g. symbiosis and phase variation (Forst and Nealson, 1996; (Bai et al., 2013). Recently, the genome sequences of five different species of Steinernema, were analysed and a common set of genes that are probably involved with parasitism were identified (Dillman, et al., 2015) Selective breeding Artificial selection is an effective method used to enhance biocontrol traits (Hoy, 1985), including heat resistance and movement in cold temperature, both of which are controlled by genetic and environmental factors (Ehlers et al., 2005). Sufficient heritability is required to achieve improvement via directed selection. If the trait of interest has low heritability, then the chances of success are diminished relative to phenotypes with high heritability (Burnell, 2002). The inherent genetic variation within a population is also important to the success of any selective breeding procedures. For example, due to low genetic diversity it is not possible to increase the ultraviolet tolerance of S. feltiae, whereas host-finding ability has been improved by the selective breeding process, due to sufficient genetic variation underlying this trait (Gaugler et al., 1989). Selective breeding of H. bacteriophora has improved and stabilized characteristics that enhance production in liquid culture (Anbesse et al., 2013). In some studies this improvement was achieved only when the selection procedure was combined with an adaptation process. In these cases, the adaptation process provided a greater phenotypic 16

30 variation in the populations that concluded a success in selective breeding. Through a desiccation tolerance trial, without the adaptation process, IJs were more susceptible against todesiccation stress compared to those that passed the adaptation process, (Strauch et al., 2004). The phenotypes of selected strains tend to gradually revert towards the unselected state, whenever the artificial selection pressures are removed (Hastings, 1994; Burnell, 2002). Cryopreservation of the selected nematode lines and reapplication of selection criteria to the selected strain after a certain period of time can preserve enhanced traits within a population (Burnell, 2002). To enhance the improvement process further, genetic selection can be combined with hybridization (Mukuka et al., 2010a, b) Hybridization The transfer of beneficial traits from one strain to another can be achieved through hybridization (Shapiro et al., 1997; Shapiro-Ilan et al., 2005a). In one of the initial investigations into hybridization, Shapiro et al. (1997) crossed a commercial strain of H. bacteriophora (HP88) and a heat tolerant strain (IS5). The hybrid nature of the progeny was confirmed using a marker mutant of the HP88 strain (Hp-dpy-2) and by backcrossing (Shapiro et al., 1997). The hybrid nematodes after serial culturing through the G. mellonella, three and six passages, exhibited a significantly higher heat tolerance characteristic than the commercial strain and was comparable to the local strain (Shapiro et al., 1997). Hybridization can be used to incorporate beneficial traits from EPNs that have been recovered from diverse geographic regions that, on their own, might not possess all of the characteristics that are needed for successful commercialization. Following hybridization in 17

31 the laboratory, field release can occur where natural selection could potentially result in the formation of a dominant strain with better efficacy (Legner, 1972). It has been suggested that this process could be used in a pre-adapted hybrid strain of S. feltiae to increase resistance against desiccation (Nimkingrat et al., 2013). The heat tolerance of a hybrid strain of H. bacteriophora increased from 38.5 o C to 39.2 o C after adaptation (Ehlers et al., 2005). Also, when heat tolerant H. bacteriophora hybrids were cross bred with desiccation-resistant parents, improvements in two traits, i.e., heat and desiccation tolerance, were seen in the progeny (Mukuka et al., 2010a) Mutagenesis Mutagenesis can be utilized to induce variation in the genome; however only one or a small number of genes can be modified each time (Burnell and Dowds., 1996). Mutagenesis commonly results in the deactivation of genes and can lead to detrimental effects rather than enhancement of positive traits (Burnell and Dowds, 1996). The mutagenesis process can be used to create a foundation to improve EPN efficacy, such as in S. feltiae where two mutant groups demonstrated increased capacity for vertical travel in the soil and increased penetration of the pest body compared to the original population (Tomalak, 2006) Discovery of new strains or species The use of EPNs for pest control could be improved or expanded by finding new species or strains that contain superior levels of desirable characteristics such as virulence, environmental competence or reproductive capacity (Grewal et al., 2001; Wang et al., 2007; Shapiro-Ilan et al., 2012). For example, in a lab trial, a newly discovered strain of S. riobrave 18

32 caused a significant increase in mortality of subterranean termites compared to a commercial sample (Yu et al., 2010). However, the discovery of new strains and species does not always lead to increased efficacy, such as the newly discovered species, H. mexicana, which did not demonstrate increased virulence against a variety of pest larvae compared with well-known species including H. bacteriophora, H. megidis, and S. carpocapsae (Shapiro-Ilan et al., 2005b). Furthermore, virulence is only trait that must be considered in the commercialization of EPNs. Strains which are readily produced in vitro may, for example, be commercialized ahead of a more virulent alternative that cannot be readily mass produced Methods of genetic engineering Genetic engineering has been considered as one possible approach to improve beneficial characteristics of biocontrol agents (Segal and Glazer, 2000). Procedures used to manipulate the genome of C. elegans and Escherichia coli (Enterobacteriales: Enterobacteriaceae), provide established methods that could be applied to modify EPN species and their symbiont bacteria (Burnell and Dowds, 1996). Studies on genetic manipulation of EPNs have focused on improving the heat resistance characteristics of the HP88 strain of H. bacteriophora. A heat-shock protein gene (hsp70 A) from C. elegans was successfully introduced into H. bacteriophora (Hashmi et al., 1998). In 1996, the first transgenic EPN, a strain of H. bacteriophora, was introduced to the environment in a field trial. A heat-shock protein gene, for improving heat resistance, was transferred from the freeliving nematode C. elegans to this nematode strain; however no advantages over the wildtype were recorded (Gaugler et al., 1997). 19

33 1.5 Trait deterioration Serial culturing, which involves the repeated culture of organisms from one generation to the next using progeny from the previous generation, frequently leads to trait deterioration (Hopper et al., 1993; Shapiro et al., 1996; Radwan et al., 2004). Trait deterioration is often inevitable for many organisms both in laboratory and industrial settings and with both in vivo and in vitro production methods (Shapiro et al., 1996). Selection for improved virulence in culturing can cause the loss of other important traits in the nematodebacteria complex, such as adaptability to the environment, reproductive capacity, and virulence (Shapiro et al., 1996; Wang and Grewal, 2002; Bai et al., 2005; Bilgrami et al., 2006). Most reports of deterioration have concentrated on the nematode partner during in vivo culture and declines have been reported in various traits, such as storage stability in S. carpocapsae (Gaugler et al., 1989), virulence and environmental tolerance (Shapiro et al., 1996), nematode fecundity, UV resistance in H. bacteriophora (Wang and Grewal, 2002) and infectivity and reproductive capacity in S. glaseri (Stuart and Gaugler, 1996). Sometimes improvements are observed following in vivo culture, such as increased virulence in H. bacteriophora when repeatedly sub-cultured in G. mellonella larvae (Wang and Grewal, 2002). In vivo culture of S. carpocapsae and H. bacteriophora has also led to the simultaneous decline of several significant traits that are crucial to biological control including virulence, heat tolerance, fecundity, host finding and tail standing, (formerly called nictation), with both the bacterial and nematode partner being implicated in trait changes (Bilgrami et al., 2006). 20

34 Pathogenic efficacy, resistance to high temperatures, fertility and host-seeking ability of H. bacteriophora could decrease during serial culturing. A reduction in beneficial traits, such as virulence, fecundity and heat tolerance, was observed during serial culture of H. bacteriophora (Bai et al., 2005; Bilgrami et al., 2006). Also, in S. carpocapsae, with the exception of host-seeking, a reduction in tail standing was detected (Bilgrami et al., 2006). The individual role of nematode and its symbiotic partner in trait deterioration by serial culturing was studied. The results concluded that changes in the bacteria were responsible for the reductions observed in most of the above-mentioned traits in both species, while changes in the nematode only had an effect on tail standing characteristics in S. carpocapsae (Bilgrami et al., 2006). 1.6 Trait stabilization The use of homozygous inbred lines was suggested as a potential mechanism that could be used to stabilize important traits in biocontrol agents (Hopper et al., 1993). The advantage of this technique is that when lines are homozygous, there is no chance for inbreeding depression or inadvertent selection. There is the possibility that some lines will be similar to a wild-type population, but others may have superior characteristics in efficacy and other beneficial traits, and, as an additional advantage, these traits are also genetically fixed (Bai et al., 2005; Chaston et al., 2011). In contrast, inbreeding throughout an entire population can lead to the depression of certain traits (e.g., specific destructive recessive alleles may become fixed) and can contribute to trait decline in EPNs (Chaston et al., 2011). The crucial reason for using inbred lines is that they have the ability to defend against the loss of beneficial traits that can occur during the serial culturing of biocontrol organisms 21

35 (Hopper et al., 1993). Therefore, a diverse array of inbred lines from the parent strain could create an opportunity for producer companies to select those lines with superior traits and propagate them on a commercial level. The hypothesis that inbred lines would deter trait deterioration in biocontrol agents was first tested in EPNs (Bai et al., 2005). The approach to creating inbred lines of EPNs is based on procedures described several decades ago for C. elegans and H. bacteriophora (Johnson and Wood, 1982; Glazer, 1991). First, surface sterilized IJs are introduced to the monoxenic bacteria (derived from the particular nematode isolate). Axenic eggs are then obtained by standing the gravid females in a stock solution 2. The eggs are then transferred to fresh plates pre-seeded with symbiotic bacteria. In heterorhabditids, hermaphrodites are selected to create the first generation of the homozygous line. These nematodes are individually relocated to new plates containing the same bacterium species, and this procedure is repeated for seven generations to create the homozygous inbred lines (Glazer et al., 1991). The genotypes of these homozygous lines are approximately 95% similar to each other (Hartl and Clark, 1989). 1.7 Cryopreservation to maintain beneficial traits Another approach to preserving beneficial traits in EPN is through cryopreservation. The use of liquid nitrogen allows for long-term storage of EPNs, e.g., the viability of cryopreserved S. carpocapsae cultures ranged from over 50 % after 8 months (Popiel and Vasquez, 1991) to up to 63% survival after three years (Curran et al., 1992). There are two stages of incubation, in a glycerol solution and methanol, before samples are submerged in liquid nitrogen, although details of optimal procedures may vary based on species and strain mol of NaOH/10% household bleach solution that is diluted 1:1 with saline water (1.2% NaCl). 22

36 (Curran et al., 1992). The IJ concentration in each step should also be considered; a high concentration of IJs typically results in a higher level of survival, up to a certain point, e.g. the highest survival of S. carpocapsae occurred with 60,000 IJs/ml. in glycerol and 25,000 IJs/ml. in Ringer s solution (Bai et al., 2004). Survival after cryopreservation could reach 80% after the initial attempt (Popiel and Vasquez, 1991), but could increase up to 100%, e.g for S. carpocapsae, with improvements in methodology and optimization of the IJ concentration (Bai et al., 2004). Cryopreservation in liquid nitrogen has been shown to be a superior technique to preserve nematodes over the long term, compared to other lab methods such as cold storage or storage at room temperature (Wang and Grewal, 2002). However, faulty lab equipment, human error, cost and the possibility of genetic bottlenecks occurring are some of the disadvantages of using cryopreservation compared to the approach of creating homozygous inbred lines (Wang and Grewal, 2002; Bilgrami et al., 2006). 1.8 Research objectives Strain development is a constant goal in the EPN industry, but once a superior biocontrol agent is obtained (through improvement methods or by discovery), the next important question is whether it can be maintained. Stability of improved strains and methods to stabilize traits are of paramount importance. The inbred line procedure that can be combined with trait improvement has shown significant promise as a means by which beneficial characteristics of EPNs can be stabilized. Stabilized lines with superior characteristics, such as virulence, reproductive capacity and 23

37 storage stability, provide a prospect for EPN producers to achieve a novel product with increased biological control efficacy. The objectives of this thesis are as follows: 1. To extract a local Canadian strain of H. bacteriophora from the soil. 2. To prevent deterioration of beneficial traits in the new strain during serial culturing by using the inbred line technique; and 3. To stabilize superior EPN lines with beneficial traits, especially virulence in colder temperatures, to an effort to enhance their potential as commercially available biological control agents. 24

38 CHAPTER TWO Creating homozygous inbred lines from a Canadian strain of Heterorhabditis bacteriophora 2.1 Abstract The use of entomopathogenic nematodes (EPNs) for insect pest management is being considered in many countries as an alternative to chemical pesticides. However, serial culturing can compromise EPN efficacy via the deterioration of beneficial traits. Creation of homozygous inbred lines has been recommended as a viable method by which negative effects of serial culturing can be minimized in EPNs (Bai et al., 2005). The current research objective was undertaken to generate superior stable lines from a Canadian strain of Heterorhabditis bacteriophora that could be used against locally destructive insect pests. Forty-one inbred lines were created in the first generation from a mixed population of four parental lines (isolated in Hanover, Ontario, Canada) based on procedures described by Johnson and Wood (1982), and Glazer et al. (1991). Only ten homozygous inbred lines successfully completed seven generations of self-reproduction. The duration of the life cycle was compared among inbred lines at room temperature. Efficacy trials to compare the relative virulence of the different inbred lines and the parental population against G. mellonella larvae were also carried out at room temperature. The life cycle comparison results indicated differences among inbred lines; inbred line 225 and 415 had the shortest and the longest duration, respectively. Also, the virulence of the inbred lines varied, ranging from 54 to 94% after 96 h after inoculation. In contrast, the wild-type parents caused 50% mortality after 96h. These results confirm that several of the inbred lines created possess 25

39 superior traits compared to the parental line, characteristics that could be relevant to their commercial development as biocontrol agents. Key words entomopathogenic nematodes, biopesticides, serial culturing; Galleria mellonella 2.2 Introduction Biocontrol of insect pests by entomopathogenic nematodes (EPNs) is an effective and safe pest management tactic. This group of nematodes, which belong to the genera Steinernema and Heterorhabditis, with their symbiont bacteria Xenorhabdus spp. and Photorhabdus spp., respectively, are capable of killing the larval stages of a wide range of insects (Poinar, 1990). Entomopathogenic nematodes can be raised by in vivo and in vitro serial culturing methods. However, long term serial culturing can result in trait deterioration. Use of the inbred line technique is recommended to eliminate or reduce damage that may result from serial culturing (Bai et al., 2005, Chaston et al., 2011). Heterorhabditis bacteriophora is one of the most favoured EPN species for commercial production and it has been reported as a reliable species for control of a wide range of soil dwelling insect pests (Shapiro-Ilan and Gaugler, n.d.). A local strain of H. bacteriophora was originally extracted from a soil sample collected in Hanover, Ontario, in September It initially demonstrated promising virulence, superior efficacy and reproductive capacity but this deteriorated significantly after in vivo culture in G. mellonella larvae for thirteen generations. Several distinctive characteristics indicated that beneficial traits of this strain diminished, including: reduced host mortality through the rearing process even though the same IJ concentration was used 26

40 throughout; and that G. mellonella cadavers turned beige instead of the normal dark-red color in successive cultures. To maintain quality of this biopesticide product, annual extraction of the isolate from soil samples was required. Therefore, the objectives of this research were to create homozygous inbred lines of a Canadian strain of H. bacteriophora and to select superior lines with stabilized beneficial characteristics to prevent trait deterioration in serial culturing. 2.3 Materials and methods Local strain extraction Sixteen soil samples were collected from several areas of a residential lawn in Goderich (latitude: 43 o N, longitude: 81 o W) and Hanover (latitude: 44 o 8 39 N, longitude: 81 o 1 26 W), Ontario, in June Samples were taken at points under the canopy line of perennial trees; soils were damp when they were collected. The farm owners confirmed that there were no historic applications of entomopathogenic nematodes at these sites. Chemicals had not been applied in the sample area. Approximately 1 kg of soil was collected at two depths (< 25 cm and cm from soil surface) at each of eight locations. All soil samples were placed in sealed bags inside an insulated Styrofoam box and were kept cool through the use of ice packs inside the cooler. Samples were shipped by express courier on the same day of collection to the laboratory at Natural Insect Control (NIC), Stevensville, Ontario, and were stored in an incubator at 7 o C upon receipt. The nematode extraction process was based on the Whitehead tray method (Whitehead and Heming, 1965). The equipment used included a small metal flat-bottom sieve, lined with a single layer of tissue paper, and placed inside a larger plastic box. A thin 27

41 layer (2 cm) with approximately 500 gm of soil was spread on top of the paper for subsequent procedures. Five-hundred millilitres of distilled water was added to the bottom of the plastic box until it reached the level of the paper and moistened the base of the soil layer. The process provided a means by which nematodes moved from the soil into the water. After 72 h the water was collected from the plastic container and placed in 15 ml centrifuge tubes (Fisherbrand, Fisher Scientific, Toronto, Ontario). Tubes were centrifuged at 5000 rpm for 2 min. The supernatant was removed by pipette and discarded, while the bottom portion containing nematodes was transferred to an empty 75 ml sterile flask. The presence of nematodes was confirmed by observing a sample of the extract under a microscope. To separate EPNs from other groups of nematodes, i.e., free-living or plant parasitic, in the samples, they were introduced to 7 th instar G. mellonella (Vanderhorst Wholesaler Inc., St. Marys, OH) using a micropipette and held at 22 C, 55 % RH for 96 h. The dead larvae were then separated and placed individually on White traps (White, 1927). White traps were made by placing an inverted 100 ml plastic Solo-cup, covered with a 9 cm diam filter paper (Reeve Angel Filter Paper Grade 202), into the center of a 500 ml plastic cup (both from GT French Paper Ltd, Niagara Falls, Ontario); distilled water (50 ml) was poured into the bottom of the larger cup, and a single dead larva was placed on the top of the moistened paper. All traps were misted and monitored daily for the presence of EPNs. After 12 days, three of the traps from a single soil sample originally collected from Hanover contained EPNs. These were subsequently identified as H. bacteriophora based on their morphological characteristics (Adams and Nguyen, 2002). All of the isolated nematodes were then pooled together in a single, sterile flask and were stored at 13 o C for later use. Their 28

42 identity was confirmed with a PCR-based assay of a subsample, sent to A and L Laboratories, London, Ontario Media preparation for bacterial culture Three types of artificial media were prepared in advance for use in the bacterial culture described below: nutrient agar, a selective culturing medium, and lipid agar. Nutrient agar media was prepared according to label instructions (VWR, Mississauga, ON). After autoclaving, (121 C, 20 mins) the medium was dispensed to 9 cm diam Petri dishes, allowed to cool and solidify under a bio-safety hood (Biobase BSC-700II, Shandong, China), and held at 4 C until use. Lipid agar was prepared by boiling 15 gm agar, 8 gm nutrient broth (Boreal Science, St. Catharines, ON), 5 g yeast extract, 4 ml corn oil, 96 ml corn syrup (these three products were obtained from local distributors), and 2.5 ml magnesium chloride x 6 hydrate (Scholar Chemistry, Boreal Science, St. Catharines, ON), in 900 ml distilled water. The medium was autoclave-sterilized as described above, and Petri plates (9 cm diam) prepared. Pre-prepared plates of T7 & TTC (Tergitol 7 agar and 2, 3, 5-triphenyl tetrazolium chloride) were obtained (Thermo Scientific, distributed by Fisher Scientific, Mississauga, ON) and used as selective control medium Inbred line development The development of inbred lines was undertaken in the laboratory at NIC following the procedures of Poinar and Thomas (1966), Johnson and Wood (1982) and Glazer et al. (1991), as described below (Fig. 2.1). 29

43 Figure 2.1. Inbred line development procedure; two steps, i.e. hermaphrodite selection and individual juveniles transfer, is repeated seven times to create homozygous population. 30

44 Inoculation. Ten healthy 7 th instar G. mellonella were inoculated by immersing them in a 1 ml suspension of distilled water containing approximately 1000 IJs of the soilextracted, wild-type H. bacteriophora for 30 sec. All the inoculated larvae were placed in a 10 cm diam sterilized Petri dish (Fisher Scientific, Mississauga, ON), lined with a single layer of filter paper. Excess inoculum was added to the Petri dish, which was held in darkness at 25 o C; inoculated larvae were used to determine the optimum time for haemolymph extraction. Haemolymph extraction. For culturing symbiont bacteria, the haemolymph extraction procedures were applied in a bio-safety hood, using sterile tools that were decontaminated by rubbing with 96% ethyl alcohol followed by an exposure to a flame. After h, Galleria mellonella larvae were rolled from side to side with lab tweezers to determine if they were infected, as infected larvae typically appeared lethargic during this process, and larvae deemed lethargic were selected for the trial. Selected larvae were surfacesterilized by immersion in medical grade ethyl alcohol for 3 sec; larvae were then washed in sterile distilled water for an additional three seconds. Larvae were dried by transferring them to a single layer of sterile filter paper. A sample of the haemolymph was then collected from the surface-sterilized larva using the following steps: i. The larva was held from the dorsal part of its body using curved forceps (Fig. 2.2A). ii. Held with the head angled slightly down and with gentle pressure applied via the forceps, the middle true leg was cut with micro scissors; the pressure caused the haemolymph to ooze through the wound (Fig. 2.2B). iii. A haemolymph sample was collected in an inoculation loop (Fig. 2.2C) and applied onto nutrient agar in a four-quadrant streak pattern (Fig. 2.2D). iv. The inoculated plates were sealed with Parafilm (Bemis, Neenah, WI, USA) and inoculated at 25 o C. 31

45 A B C D Figure 2.2. Haemolymph collection procedures. (A) The inoculated and surface-sterilized Galleria mellonella larva was held at its dorsal side with curved forceps, with gentle pressure and angling the head downwards. (B) After cutting off the middle leg, haemolymph leaks out. (C) Haemolymph is collected by a sterile inoculation loop. (D) The haemolymph is cultured on nutrient agar in a four quadrant pattern. Photographs: S. Sharifi-Far. 32

46 The optimal time to extract haemolymph and recover the bacterial symbiont during phase I (i.e. desired phase for nematode production) of the infection process has been reported to be h after inoculation (David Shapiro-Ilan, personal communication); however, in this trial, no Photorhabdus luminescens colonies were observed on plates prepared using haemolymph samples collected h after larval inoculation. It was determined that the best extraction time was 49 h after inoculation which is similar to that previously reported (e.g., Han and Ehlers, 2001). Recovery of single colonies of Photorhabdus luminescens. Single colonies of P. luminescens were removed from the nutrient agar plates after h incubation (Fig. 2.3A). Using a sterilized inoculation loop, one portion of a single colony was transferred to a new nutrient agar plate and another part of the same colony was cultured onto T7 and TTC media to distinguish between bacterial phases I and II (Fig. 2.3B). In contact with phase I P. luminescens, the colour of the T7 & TTC plates changed from green to colourless, due to dye adsorption (Fig. 2.3C). Colonies on the corresponding nutrient agar plate were orange and these cultures were selected as a source of pure bacteria for future experimental work; plates were sealed with Parafilm and held at 3 to 5 o C. When cultures were prepared, a portion of a phase I P. luminescens colony was removed with an inoculation loop and cultured on a fresh media plate. Inoculated plates were sealed with Parafilm and incubated at 25 o C for 96 h (Fig. 2.3D). Any contaminated plates were discarded. Surface sterilization of nematodes. Two 15 ml samples were taken from the IJ suspension derived from the White traps, and transferred to sterile centrifuge tubes and centrifuged at 5000 rpm for 2 min. The supernatant was removed using a sterile pipette and 10 ml of a 10% household bleach solution added to the tubes; the IJs were gently re-sus- 33

47 A B C D Figure 2.3. (A) Four quadrant streak plating of Photorhabdus luminescens on nutrient agar with a single colony of the first phase of symbiotic bacteria visible in the fourth streak zone. (B) Inoculated nutrient agar plates from single colonies (left) and their specific corresponding T7 & TTC media plates (right) (C) Dye adsorption in T7 & TTC media plate confirms that P. luminescens is in phase I. (D) Development of P. luminescens in the nutrient agar plate after one hundred and twenty hours at 25 o C. Photographs: S. Sharifi-Far. 34

48 pended in the bleach. The tubes were inverted manually for 8 min to surface-sterilize the nematodes and were then centrifuged at 5000 rpm for 2 min. Bleach solution was removed and replaced with distilled water three times and centrifuged with the same speed and duration. Following this procedure, the IJ cuticle was assumed to be free from external microbes (Poinar, 1979). However, other contaminating microbes can still remain under the sheath; therefore it is preferred to establish a monoxenic culture from eggs (Lunau et al., 1993). In this study, the surface sterilizing procedure was repeated four times; they were considered as batch 1-4. Bacterial improvement. Ideally, an improved EPN should carry symbiotic bacteria that are highly virulent, multiply rapidly in and quickly kill the host. To obtain an enhanced strain of P. luminescens, growth studies were carried out on all of the bacterial cultures derived from single colonies originally isolated from the infected G. mellonella larvae. Three fresh nutrient agar and lipid agar plates were prepared from each single colony culture, using similar amounts of inoculum for each plate, and were incubated at 25 o C for 96 h. The well spread bacteria plates, from both media were selected as improved bacteria and were used for further studies. Based on the visible observations, P. luminescens cultures generally grew more rapidly on lipid agar compared to nutrient agar (Fig. 2.4A), but both cultures from both media were used in subsequent trials to investigate whether the growth medium had any effects on the nematodes or bacteria. Seeding infective juveniles and egg extraction. The surface sterilized IJs were introduced on to nutrient and lipid agar plates previously inoculated with P. luminescens; plates were held at 25 C. Nematode development was monitored on a daily basis under a 35

49 stereo-microscope (Konus ST-30-2L, Verona, Italy). Self-fertilizing hermaphrodites were observed on the media after 5-8 days (Fig. 2.4B, C, D). The hermaphrodites and other life stages present were washed in a 10 cm diam Petri dish containing 1.2% (w/v) saline solution to a total volume of 10 ml; subsequently this suspension was transferred in to a 15 ml centrifuge tube. Tubes were centrifuged at 5000 rpm for 2 min. The supernatant was removed by pipette and discarded. Then, 5 ml of stock solution (containing 0.8 g sodium hydroxide, 6.7 ml bleach and 43.3 ml distilled water) was diluted with 5 ml of distilled water and added to the centrifuge tubes. The nematode pellet was gently re-suspended and the tube inverted frequently for 9 minutes to soften the nematode body wall (Bai et al., 2013). Tubes were then centrifuged at 5000 rpm for 2 min. This caused the nematodes to burst and eggs were released from the hermaphrodites. The supernatant was removed and the eggs washed 3 times in sterile distilled water through repeated suspend-centrifuge cycles. The extracted eggs were then placed onto fresh bacterial cultures prepared on nutrient and lipid agar media. The inoculated plates were sealed with Parafilm and were stored at 25 o C. After approximately 2 to 3 days, from a monoxenic culture, hatched juveniles were washed from the dishes in 1.2% saline solution and collected in a 10 cm diam sterile Petri dish. A total of 300 juveniles, comprised of 75 individuals taken from each of four batches/replications in ca.10µl saline solution, were transferred individually onto 60 mm diam Petri dish cultures of P. luminescens in nutrient and lipid agar, to create the first generation of monoxenic parents. These inoculated plates were also sealed with Parafilm and held at 25 o C. 36

50 A B C D Figure 2.4. (A) Comparison of Photorhabdus luminescens development on nutrient agar (left) and lipid agar (right). (B) Heterorhabditis bacteriophora hermaphrodites on lipid agar plates (40x magnification). (C) Lipid agar plates with hermaphrodites, five days after introduction of surface-sterilized infective juveniles. (D) A centrifuge tube containing washed hermaphrodites before eggs were extracted. Photographs: S. Sharifi-Far. 37

51 Creating a homozygous population. Juveniles developed into male, female and hermaphrodite adults from 7 to 10 days. Subsequently, a total of 41 hermaphrodites were recovered (10 each from batches 1-3, 11 from batch 4) and constituted a parent line. At 5-6 days, 10 juveniles from each line were introduced individually onto prepared bacterial plates. These plates from both nutrient and lipid agar were inoculated with enhanced bacteria. The entire procedure (i.e. isolating the individual nematodes onto bacterial lawns and collecting the next generation of juveniles) was repeated until the 7 th generation was obtained. This generation is considered at least 95% homozygous (Hartl and Clark, 1989) and was considered to be an inbred line. In this study, 10 lines were successfully prepared; whereas 30 lines were lost at different stages of the procedure. The last generation of each line produced IJs when their nutrient source is depleted. These IJs of the 7 th generation were washed in 1.2% saline solution and an inoculum containing 1000 IJs/ml was prepared. Twenty G. mellonella larvae were inoculated by immersing the larvae in 2 ml of inoculum in a 100 ml Solo cup for 30 sec. The IJs recovered from the larval cadavers were designated as a new inbred line of H. bacteriophora. The sequence of numeration was designed by three digit numbers, where the first number represented the parental line (1-4), the second number the media type (1 for nutrient agar, 2 for lipid agar), and the third the replication (e.g. line 315 means the third parental line which was introduced to nutrient agar on the fifth replication) Duration of the inbred lines life cycle The duration of the inbred line life cycles were recorded to identify differences in development times among the lines. The average time taken for nematodes to develop from 38

52 one hermaphrodite generation to the next on lipid agar media was recorded as a life cycle for each line. Nematode development was observed under a dissecting microscope at 40x magnification. Life cycle duration for the wild-type parents, including non-cultured and subcultured batches was determined as an average 5.4 and 6.3 days, respectively. In order to clarify different wild-type parents throughout the entire study, it should be noted that non-cultured refers to passages that were limited to a single passage in parallel with other populations in order to standardize IJ age prior to bioassays; otherwise the noncultured population was not reproduced during the study Efficacy of inbred lines To assess differences in virulence among the new inbred lines after seven generations, a randomized complete block design experiment, with 12 treatments and five replications (blocks), was carried out. Treatments consisted of: non-cultured, which was the original wild-type population after soil extraction, sub-cultured that was created from the original population after 15 passages of serial culturing through G. mellonella and ten inbred lines, which were the final outcome of the inbred line process. Each experimental unit contained ten 7 th instar G. mellonella. All the original populations of each inbred line and their wild-type parents were stored in flasks with a concentration of 1000 IJs/ml at 13 o C and IJs were cultured 3 weeks before the efficacy trial. The cadavers of insects that were successfully infected were red in colour; four days after inoculation, these were placed in White traps for 7 to 10 days and IJs that emerged were stored in a vented, sterile 250 ml culture flask at 13 o C for 7 to 10 days prior to the start of the trial. Therefore, all lines and control populations were produced in the same way prior to the assay, to remove IJ age as a significant variable that could influence 39

53 the outcome of the experiment. Inoculation for each unit was achieved by submerging the G. mellonella larvae in the nematode suspension of 1 ml, at a concentration of 1000 IJs/ml for duration of thirty seconds. The inoculated larvae and remaining suspension were then added to a Petri dish (100 x 15 mm) lined with a Whatman # 202 filter paper, and the lids were secured. The Petri dishes were randomly distributed in a complete block pattern, across lab trays and were held in the dark at 22 o C for 96 h. The efficacy of the respective treatments was assessed by counting the number of cadavers which had a pink/red colouration, 4 days after inoculation. Uninfected larvae that were alive (white) or dead (black or beige) were discarded. All statistical analyses for both trials were conducted with SAS 9.3 software, at α = A generalized linear mixed model variance analysis was conducted using the GLIMMIX procedure, followed by means separations using Tukey s test. Estimates and standard error were converted to the data scale using the ilink option. Plots of studentized residuals were used to confirm the analysis assumptions. Although, a beta distribution, with logit link using the Laplace approximation was fit only for room temperature trial. 2.4 Results Inbred line development. During development of inbred lines, some distinctive characteristics were observed within particular lines. All bacterial lines grew successfully on both media types. However, in general, bacterial development was more rapid on the lipid agar than nutrient agar. Based on observations under the microscope, inbred line 324 had a characteristic hermaphrodite stage and their physical activity was relatively greater than that of the other lines. Moreover, some plates of line 421 initially carried a high number of IJs, but these rapidly died. The IJs derived from inbred line 415 were unique in that they showed 40

54 a tendency to form clumps. Uncharacteristically large IJs were observed from two plates of inbred line 225. Thirty-one of the lines died out before they reached the pre-defined endpoint of the trial. Life cycle trial. Significant differences in developmental times were observed between the inbred lines (F = 12.46, df = 9, 81, P = <.0001). Inbred line 225 had a significantly shorter life cycle than lines 123, 222, 313, 421, 424, 125 and 415. The life cycle of line 315 was significantly shorter than that of lines 313, 421, 424, 125 and 415. There were no differences among the life cycle times of lines 324, 123, 222, 313, 421, 424, 125; however, all of these lines had a significantly shorter life cycle than inbred line 415 (Fig. 2.5). Efficacy of inbred lines at room temperature. Significant differences in efficacy were observed among the lines (F = 3.99, df = 11, 44, P = ). None of the inbred lines were superior to the non-cultured wild-type parental population. However, inbred lines 421, 324, 125, 222 and 123 had superior efficacy to the sub-cultured population and inbred line 415 (Fig. 2.6). 2.5 Discussion The main objective of this research was to create a series of homozygous inbred lines from a Canadian strain of H. bacteriophora. Based on previous research, if inbred lines can be successfully produced, these lines will be genetically homogenous (Hartl and Clark, 1989); as a result, the lines would be expected to be genetically stable and their virulence would not be adversely affected by serial culturing (Bai et al., 2005; Chaston et al., 2011). 41

55 Line 123 Line 125 Line 222 Line 225 Line 313 Line 315 Line 324 Line 415 Line 421 Line 424 Life cycle duration (day) 8 a bc b bc b b bcd b cd d Inbred lines Figure 2.5. Mean duration of life cycle in days (± SE) of a local strain of Heterorhabditis bacteriophora in lipid agar media at room temperature (22 o C). The appearance of hermaphrodite in two consecutive generations were considered as the life cycle of the inbred lines. Columns with the same letter are not significantly different (P 0.05). 42

56 Line 123 Line 125 Line 222 Line 225 Line 313 Line 315 Line 324 Line 415 Line 421 Line 424 Sub-cultured Non-cultured Efficacy % a a a ab ab ab a b a ab b ab Treatments Figure 2.6. Mean percent (± SE) efficacy of homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, compared to non-cultured and sub-cultured populations of their wild-type parents, against final (7th) instar Galleria mellonella at 22 o C. The sub-cultured population was derived after 15 passages through G. mellonella, while the noncultured population was the original population collected after soil extraction. Bars with the same letter are not significantly different (P 0.05). 43

57 Several phenotypic differences have been reported among inbred lines that were created from a strain of H. bacteriophora including; heat, UV or dehydration tolerance (Glazer et al., 1991). However, no differences in the genetic heterogeneity was observed between inbred lines that were created from a H. bacteriophora strain maintained in the laboratory for over 10 years, and inbred lines that were developed from a new strain of the same species that was isolated from the field (Shapiro et al., 1997). This limited genetic difference could be related to the hermaphroditism phenomenon in this genus (Downes and Griffin, 1996). This would confirm the long-term genetic stability of the lines, created in the study. The differences in efficacy observed in this study indicate that the inbred lines that were created are genetically dissimilar. Five superior inbred lines were identified (i.e. more virulent against G. mellonella larvae than the wild-type parental population) and at least one other inbred line. These results support the findings of Bai et al. (2005), who showed that serial culturing diminished beneficial traits of the wild-type but not of the inbred lines of nematodes. Throughout the following chapters, comparison among different characteristics of inbred lines and their wild-type parents (e.g., reproduction, cold tolerance, stability in storage, virulence and the impact of serial culturing on trait deterioration) is presented. 44

58 CHAPTER THREE The impact of serial culturing on fitness and production capacity of entomopathogenic nematodes: inbred lines vs. wild-type populations 3.1 Abstract Several quantitative and qualitative characteristics (e.g. reproductive capacity, efficacy, shelf life) of a wild-type population of a local strain of Heterorhabditis bacteriophora and two commercial strains were compared with 10 newly created homozygous inbred lines to determine whether the inbred line technique resulted in nematode lines showing superior traits relevant to their commercial production and use, and generated a genetically stable population in which beneficial traits were maintained. In this series of trials, reproductive capacity and storage stability of inbred lines were compared with their original parents. An additional trial was conducted in order to investigate whether serial culturing affected efficacy and production over the culturing period. Significant differences in reproductive capacity were found among lines, with an inbred line (i.e. line 421) having the highest reproductive capacity in both small and large-scale trials, while commercial line 1 and inbred line 125 had the lowest reproductive capacity in the small and large-scale trials, respectively. Inbred line 324 had superior storage stability, while the sub-cultured wild-type parents had inferior storage characteristics. The dominance of some inbred lines (e.g. line 324) in comparison with other inbred lines and the wild-type parents was demonstrated throughout the serial culturing trial, while other inbred lines (e.g. line 415) were found to be inferior in virulence and reproductive capacity. However, detrimental effects of serial 45

59 culturing on efficacy and production were found to be reduced in the majority of created inbred lines compared to the wild-type population. Key words Heterorhabditis bacteriophora, homozygous inbred lines, virulence, storage stability, production ratio. 3.2 Introduction Entomopathogenic nematodes (EPNs) are a valuable pest management tool and have been reported to be virulent a wide range of pest species (Poinar, 1983). Both quantitative and qualitative characteristics of EPNs can significantly affect their performance as biological control agents, and variation in factors such as virulence, reproductive capacity and environmental tolerance has been extensively studied (Shapiro- Ilan et al., 2002; Shapiro-Ilan et al., 2003; Shapiro-Ilan et al., 2006; Shapiro-Ilan et al., 2012; Shapiro-Ilan et al., 2015). Maintaining desired traits and protecting them from deterioration is one of the most challenging tasks in commercial production of EPNs. Repeated use of the same strain of a wild-type population through serial culturing can jeopardize the expression of traits that are essential to their mass production, shelf life and field performance (Shapiro-Ilan et al., 1996; Wang and Grewal, 2002; Bai et al., 2005; Bilgrami et al., 2006; Chaston et al., 2011). However, the creation of homozygous inbred lines that are genetically stable can be an effective method to eliminate or reduce the impact of serial culturing (Bai et al., 2005; Chaston et al., 2011). 46

60 Small-scale production of EPNs, such as in a small White trap (White, 1927), is simpler and much easier to modify compared to a commercial production scale. In this situation moisture, humidity and temperature can be accurately controlled to provide precise results on nematode response. In large commercial production scale, EPNs are not held in optimum conditions at all times and are subjected to fluctuations in temperature, moisture and humidity. As such, it is essential to evaluate the adaptability of inbred lines to larger scale commercial production techniques. The objectives were to use inbred techniques to create genetically stabilized homozygous lines that are resistant against trait deterioration (Bai et al., 2005); then, through a series of trials, investigate selected qualitative and quantitative characteristics to identify superior lines that could be commercialized. Of specific interest, was the creation of homozygous inbred lines with improved virulence and field performance, superior reproductive capacity and stability in storage over wild-type populations. Adaptation to large-scale commercial production techniques was then evaluated. 3.3 Materials and methods Small-scale production trial To assess differences in reproductive capacity among created inbred lines, a randomized complete block design experiment was conducted, with 14 treatments and five replications (blocks) of each treatment. Treatments were: ten inbred lines; the non-cultured which was the original wild-type population after soil extraction; a sub-cultured wild-type population (i.e. original parents after 15 passages of serial culturing through Galleria 47

61 mellonella); and two commercially available H. bacteriophora samples, Commercial 1 and Commercial 2. Each experimental unit consisted of five 7 th instar G. mellonella. To generate sufficient, fresh inoculum for this trial, all treatments were cultured twice through G. mellonella, with the last round applied 21 days before the trial began. Emerged infective juveniles (IJs) from all treatments including the inbred lines, wild-type parents and commercial strains, were stored in flasks with a concentration of 1000 IJs/ml at 13 o C. Therefore, all treatments were cultured in parallel before the assay to remove IJ age or formulation as a significant factor. For the inoculation process, 10 seventh instar G. mellonella (Vanderhorst Wholesaler Inc., St. Marys, OH), were inoculated with 1000 IJs in 1 ml of distilled water for each experimental unit. The larvae were immersed in the suspension in a 100 ml Solo cup (GT French Paper Ltd, Niagara Falls, ON) for 30 sec, and were then transferred to a Petri dish (100 x 15 mm) lined with a filter paper (Reeve Angel Grade 202). The excess suspension was evenly poured into the Petri dish and then stored at room temperature (~22 o C) in the dark. Infected larvae were characterized by a change of color, from white to reddish-pink, which is characteristic in insects killed by toxins produced by the nematode s symbiotic bacterium Photorhabdus luminescens (Blackburn et al., 2005; Rodou et al., 2010). To determine the reproductive capacity of the nematode treatments a White trap was assembled using an inverted 100 ml Solo cup covered with a layer of the same filter paper was placed inside a 500 ml plastic container (GT French Paper Ltd, Niagara Falls, ON), and five infected cadavers were arranged evenly on the cup. Subsequently, 100 ml of distilled water was added to the larger container. For each individual experimental block, cadavers of similar size and color were chosen and placed on 48

62 the White trap in order to reduce any bias based on host size. Treatment efficacy was recorded by counting infected and non-infected larvae. To avoid contamination, all traps were covered with a double layer of cheesecloth which was held in place by a rubber band. Traps were held in the laboratory at 22 o C and hand-sprayed with distilled water on a daily basis. Harvested IJs were collected into aerated containers using an air pump and an air stone. Final yield counts were taken 30 days after inoculation. After 30 days, the entire suspension harvested from each replicate treatment was poured in to a 1000 ml graduated cylinder, and total volume adjusted to 500 ml. After vigorously stirring the suspension, three 0.1 ml samples were extracted from each container using a micro-pipette and live nematodes were counted under a stereo-microscope at 40x magnification. Damaged or dead EPNs were disregarded. The pipette tip was exchanged after each sample, to avoid any cross contamination. To provide a precise count of nematodes, a narrow pattern of suspension was created inside a small Solo cup lid, so that the full width of the sample could be observed in a single viewing. The entire sample was counted from left to right and the total number recorded. This procedure was repeated from right to left, and if both counts were the same, it was recorded as the final harvest of nematodes. If counts differed, a third count was performed. If the nematode suspension had a high concentration of IJs (>2000 IJs/ml), the sample was diluted prior to counting so that a more precise count could be made. The total yield of each experimental unit was calculated as the average of three samples multiplied by the total suspension volume. 49

63 3.3.2 Large-scale production trial In order to determine whether the use of homozygous inbred lines would enhance large-scale production, each treatment was assessed in a commercial production system. Treatments and methods used to maintain the starter inoculum were as described in section A total of 14 treatments were evaluated, with three replicates of each. Treatments were arranged in a randomized complete block design to minimize any effects of location on performance. For each treatment, 210 ml of inoculum was prepared, containing 1430 IJs/ml. Three replicate inoculations were carried out for each treatment; three batches of G. mellonella larvae were inoculated in individual arenas which consisted of plastic containers (L 60 x W 50 x H 20 cm) (Rubbermaid Solutions, Burlington, ON) that had been lined with a double layer of absorbent paper (GT French Paper Ltd, Niagara Falls, ON). One thousand G. mellonella larvae were introduced into each container (total 3,000 per EPN treatment) and inoculated with 70ml of EPN suspension. Lids were placed on top of each container, which were held in the dark at 25 o C for 5 h. At 5 h the lid was opened slightly (i.e. 2 cm) for ventilation. Ninety-six hours after inoculation, 1,500 infected hosts were randomly chosen (ca. 500 per replicate) for each EPN treatment and were divided among three 75 L containers (Gracious Living TM, Solutions, Burlington, ON) (L 107 x W 42 x H 16 cm), as three experimental units. A wooden frame (L 86 x W 35 x H 2.5 cm) was inserted inside the container and a piece of plastic fencing (L 102 x W 32 cm) was placed on top of each frame. The plastic fencing material was covered with a double layer of grade ten cheesecloth to 50

64 maintain moisture for the cadavers during the manufacturing process. All units were sprayed with approximately 100 ml of distilled water twice a day. All cadavers were transferred on the same date and each replicate (block) was kept in a specific area of the production laboratory. The experimental units for each replicate were distributed randomly on a production bench. Constant humidity (55% RH) and temperature (25 o C) were maintained inside the production laboratory. Three oscillating fans continually circulated air so that conditions were similar throughout the entire room. Fly traps and sticky strips were installed in different areas to avoid any fly infestation. Twelve days after inoculation, the first emerged nematodes were harvested from the production units and stored in a separate bucket which was aerated by an air-pump in an incubator at 13 o C. Collection of emerged nematodes continued for up to 28 days after inoculation. To determine IJ production, the same protocols as those used in the small-scale production trial were applied, except that five samples were counted to obtain an estimate of yield Storage stability trial The goal of this trial was to determine if it is possible to improve the storage stability of nematodes through the inbred line procedure. To evaluate differences in storage stability among commercial lines, new inbred lines and their wild-type populations, a total of fourteen treatments were included in the trial. The trial was set up as a randomized complete block design with five replicates (blocks) per treatment. Treatments were the same as those identified in the small-scale trial, and inoculum was maintained under similar conditions. 51

65 Infective juveniles produced in the large-scale production trial were used to evaluate shelf life. A composite EPN sample was prepared from the three production replicates of each treatment. The sample was thoroughly mixed and divided equally into five 10 litre plastic containers (L 22 x W 19 x H 30 cm; approx. 5 L per container), with the concentration of < 5000 IJs/ml, twenty-one days after inoculation, although for a more accurate calculation, the fourteenth day after inoculation (i.e. peak of production) was specified as the first day of storage. The contents of each container were aerated using an air pump with an aquarium air stone (Top Fin, Pet Smart, Niagara Falls, ON) and were stored at 13 o C. Samples were taken weekly to assess nematode viability. After mixing the nematode suspension evenly, three 0.1 ml. samples were taken from each of the five containers per treatment. Viability was calculated based on the total number of live and dead nematodes in the samples. As no differences were observed in IJ mortality over the first 6 weeks, samples were collected every 7days to week 7, and every 15 days, thereafter Serial culturing trial To measure the impact of serial culturing on efficacy and reproductive capacity of the new inbred lines, a total of twelve treatments were included in the trial. The trial was set up as a randomized complete block design, with twelve treatments and five replications (blocks) per treatment. Treatments consisted of ten inbred lines, the non-cultured, which was the original wild-type population after soil extraction, and the sub-cultured wild-type population (i.e. the original parents after fifteen passages through G. mellonella). To produce a fresh starter inoculum for the trial, five batches of 10 healthy 7 th instar G. mellonella were immersed in 1 ml of second passages EPN suspension with concentration 52

66 of 1,000 IJs (100 IJs/larva) for 30 sec., in a 100 ml Solo cup. The inoculum was held in a flask at 13 o C. This was repeated for each treatment included in the trial. The inoculated larvae and remaining suspension were transferred to a Petri dish (100 x 15 mm) lined with a single layer of filter paper (Reeve Angel Filter Paper Grade 202). The dishes were kept in the dark at 22 o C. Ninety-six hours after inoculation, 25 infected cadavers from each treatment (i.e. 5 from each replicate) were selected and placed in five White traps, five G. mellonella cadavers per each trap. The White traps were set up as described above for the small-scale production trial. In fact, the nematodes derived from the small-scale production trial were used for the first series of serial culturing reproductive capacity. All traps were sprayed daily with distilled water. In most cases, the first series of emerged nematodes started twelve to fourteen days after inoculation. Nematodes harvested from the small-scale production trial were stored under aeration for seven days and were then used to inoculate fresh batches of G. mellonella larvae using previously described protocols (Section 3.3.1). Nematodes harvested after each serial passage were used to prepare inoculum for the next passage by making a composite sample of IJs (i.e. 5 ml of collected nematodes from each White trap per treatment). This was diluted to obtain a concentration of 1000 IJs/ml. Fifty 7 th instar G. mellonella were immersed in five ml of the inoculum suspension for 30 sec.; batches of 10 treated larvae were transferred into each of five Petri dishes (100 x 15 mm) and were incubated under conditions as described previously. This process was repeated for fifteen passages. The efficacy of each passage was measured by evaluating the pink/red coloration of cadavers, four days after inoculation. Reproductive capacity was assessed by counting the 53

67 number of IJs that emerged from the infected G. mellonella cadavers placed on the White traps over three weeks; however, only counts taken after the first, fifth, tenth and fifteenth passages were considered for statistical analysis. Peak production of IJs typically occurred ten to fourteen days after inoculation, and in most treatments diminished after seventeen days. The only exception was for inbred line 415 which required a seven day extension for harvesting. Consequently, when this line completed its eleventh passage, all other lines had completed their fifteenth passage Statistical analyses All statistical analyses were conducted with SAS 9.3 software, at α = A generalized linear mixed model variance analysis was conducted using the GLIMMIX procedure, followed by a multiple means comparison on the model scale that was performed using Tukey s adjustment. Estimates and standard errors were converted to the data scale using the ilink option. Plots of studentized residuals were used to confirm the analysis assumptions. For qualitative trials (i.e. mortality assessment in the storage stability and serial culturing trials), a beta distribution was fitted to the data with logit link using the Laplace approximation. However, for all quantitative trials (i.e. reproductive capacity in the small, large and serial culturing trials), the Laplace and beta distribution options were not required and a normal identity link analysis was carried out. In the storage stability trial, data from day 65 was missing for one replicate each of inbred lines 123 and 415 due to a malfunction of the air pump malfunction; and data at day 80 was missing for one replicate of the non-cultured treatment due to a clogged air stone. The SAS program estimated the missing data values for all treatments in both observations. The impact of serial culturing on efficacy of each individual treatment over time was analysed. 54

68 Efficacy differences within passage were evaluated with multiple comparisons using the Tukey-Kramer grouping test. Effects of serial culturing on the reproductive capacity within each treatment were assessed using SAS software with the GLIMMIX procedure, restricted maximum likelihood estimation technique. Data were adjusted using Restricted Maximum Likelihood, to allow differences in reproductive capacity by passage to be compared using the Tukey-Kramer grouping test at α= Results Small-scale trial There were significant differences in reproductive capacity among treatments (F = 5.85, df = 13, 52, P < ). The superior treatments in this trial were inbred lines 222, 315, 421 and 123, which had significantly higher reproductive capacity than inbred line 415 and commercial product 1 (Fig. 3.1). Inbred lines 313, 225, 324, non-cultured wild-type population, commercial product 2, and inbred lines 125 and 424 had a higher reproductive capacity than commercial product 1. However, there were no differences among the calculated reproductive capacities for the sub-cultured wild-type population, inbred line 415 and commercial product Large-scale trial Significant differences in reproductive capacity were observed between treatments in the large-scale trial (F = 3.07, df = 13, 26, P = ). The reproductive capacity of inbred 55

69 Figure 3.1. Mean (± SE) reproductive capacity per Galleria mellonella cadaver in the small-scale production trial. The trial was run at 22 o C, and included ten homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, and two commercial strains. The sub-cultured population went through 15 serial passages in G. mellonella, while the non-cultured population was the original population collected after soil extraction. Bars with the same letter are not significantly different (P 0.05). 56

70 line 421 was higher than that of inbred line 125, however no other differences among treatments were observed (Fig. 3.2) Storage stability trial Significant differences in mortality in this trial were demonstrated in figure 3.3. These differences among treatments after 50 days of storage were significant (F = 8.76, df = 13, 52, P < ). Mortality of inbred line 324 was significantly lower than inbred lines 123, 125, 222, 225, 315, 415, commercial product 2, and the sub-cultured (Fig. 3.3A). Also, mortality of the non-cultured wild-type population was significantly lower than inbred lines 222, 225 and 415. Mortality was significantly lower for inbred lines 123, 125, 313, 421 and 424, commercial product 1, and commercial product 2, compared to inbred lines 222 and 225. The mortality of inbred line 315 was also significantly lower than inbred line 225, but there were no differences among the sub-cultured population, and inbred lines 222, 225 and 415. After 65 days of storage, significant differences in mortality were observed among treatments (F = 12.92, df =13, 50, P < ). Significantly lower mortality was observed for inbred line 324 than inbred lines 125, 222, 225, 313, 315, 415, 424, commercial product 1, commercial product 2, and the sub-cultured population (Fig. 3.3B). 57

71 Figure 3.2. Mean (± SE) reproductive capacity per Galleria mellonella cadaver in the large-scale production trial. The trial was run at 25 o C, and included ten homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, and two commercial strains. The sub-cultured population went through 15 serial passages in G. mellonella, while the non-cultured population was the original population collected after soil extraction. Bars with the same letter are not significantly different (P 0.05). 58

72 Line 123 Line 125 Line 222 Line 225 Line 313 Line 315 Line 324 Line 415 Line 421 Line 424 sub-cultured non-cultured Com (1) Com (2) Mortality % h gh fgh efgh defg defg cde def ab abc abcd bcde a abc Mortality % e de cde cde bcd bcd bc bcd bc ab ab ab ab a e Mortality % de cd cde cde cd cde cde cd bcd abc abcd ab a A B C Treatments Figure 3.3. Mean (± SE) % mortality of homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora compared to non-cultured and sub-cultured populations of their wild-type parents, and two commercial strains. All IJ suspensions were aerated and stored at 13 o C for A) 50, B) 65, and C) 80 days. Bars within a figure with the same letter are not significantly different (P 0.05). 59

73 Inbred line 421, the non-cultured population and inbred line 123 had significantly lower levels of mortality than commercial product 2, inbred lines 125, 222 and 225, and the sub-cultured population. Mortality was significantly lower for inbred lines 313, 315, 415 and 424, and commercial product 1 than inbred line 222. There were no differences in mortality among commercial product 2, inbred lines 125, 222, 225 and the sub-cultured population. Significant differences in mortality after 80 days of storage were also observed among treatments (F = 18.29, df = 13, 49, P < ) (Fig. 3.3C). The inbred line 324 demonstrated the lowest mortality levels followed by inbred line 421. The sub-cultured population showed the highest mortality followed by commercial product 2 and inbred line 415. In fact, there were no significant differences in mortality levels among inbred lines 125, 222 and 415, commercial product 2 and the sub-cultured population. Storage stability over time was also evaluated within treatments. In eight of the treatments the percent mortality increased significantly after 42 days (i.e., mortality at 50 < 65 < 80 days): inbred lines 125 (F = 52.21, df = 2, 8, P < ), 222 (F = 25.65, df = 2, 8, P = ), 324 (F = 67.45, df = 2, 8, P < ), 415 (F = 114.9, df = 2, 6, P < ), 424 (F = 23.93, df = 2, 8, P = ), sub-cultured population (F = 35.84, df = 2, 8, P < ), commercial product 1 (F = 26.04, df = 2, 8, P = ), and commercial product 2 (F = 77.7, df = 2, 8, P < ). For inbred lines 123 (F = 19.64, df = 2, 6, P = ) and 315 (F = 20.15, df = 2, 8, P = ), mortality did not significantly differ between day 50 and day 65, but was significantly lower at day 80. In inbred lines 225 (F = 5.34, df = 2, 8, P = ) and 421 (F = 6.46, df = 2, 8, P = ), mortality was significantly higher at day 80 than day 50, but mortality at day 65 did not differ significantly from the other time periods. For inbred line 313 (F = 20.53, df = 2, 8, P = ) and the non-cultured 60

74 population (F = 17.59, df = 2, 7, P = ), mortality at day 50 was significantly lower than at day 65 and 80, but mortality did not significantly differ between the two later dates Serial culturing trial The efficacy results against G. mellonella for the entire trial are illustrated in figure 3.4. The significant differences were observed among treatments after the first passage (F = 3.99, df = 11, 44, P = ). Inbred lines 123, 125, 222, 324 and 421 showed a higher level of efficacy than inbred line 415 and the sub-cultured population (Fig. 3.4A). There were no differences in efficacy among the non-cultured population, inbred lines 225, 313, 315, 415 and 424, and sub-cultured population. After five passages, significant differences in efficacy were observed among treatments (F = 5.01, df = 11, 44, P < ). The inbred line 415 and sub-cultured population had lower virulence than inbred lines 123, 125, 313, 324, 421 and 424, and noncultured population (Fig. 3.4B). Virulence of the sub-cultured population was also lower than for inbred line 222. There were no differences in virulence among inbred lines 225, 315 and 415, and the sub-cultured population. Differences in virulence among treatments (α=0.05) was higher after the tenth passage (F = 7.86, df = 11, 44, P < ). Inbred line 324 was superior to many others tested; efficacy was significantly higher than for inbred lines 313, 415, 421 and 424, and the non-cultured and sub-cultured parental lines. The virulence of lines 123, 125, 222, 225, and 315, was greater than line 415 and the sub-cultured parental line. Also, the efficacy of inbred lines 313, 421 and 424, and the noncultured parental line was significantly better than the sub-cultured parental line. There were no differences between inbred line 415 and the sub-cultured parental line (Fig. 3.4C). 61

75 Figure 3.4. Effect of serial culturing on the virulence (mean ± SE percent efficacy) of 10 homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, against Galleria mellonella larvae after the A) first, B) fifth, C) tenth, and D) fifteenth passage through G. mellonella at 22 o C. Bars within a figure with the same letter are not significantly different (P 0.05). 62

76 After the eleventh passage, G. mellonella cadavers in the sub-cultured parental line treatment did not turn pink/red; therefore this treatment was withdrawn from subsequent stages of the experiment, and was thus not included in subsequent comparative analyses. Significant differences in efficacy among treatments were observed after the fifteenth passage (F = 6.45, df = 10, 40, P < ). Inbred line 324 was more virulent than inbred lines 313, 415, 421 and 424, and the non-cultured parental line (Fig. 3.4D). Inbred lines 123 and 222 were more efficacious than inbred line 415 and the non-cultured parental line. The efficacy of inbred lines 125, 225 and 315 was higher than the non-cultured parental line. There were no significant differences observed among inbred lines 313, 415, 421 and 424, and the non-cultured parental line. No reduction in efficacy as a result of serial culturing was observed in six inbred lines: lines 222 (F = 1.44, df = 3, 12, P = 0.28), 225 (F = 0.92, df = 3, 12, P = ), 315 (F = 2.85, df = 3, 12, P = ), 324 (F = 0.58, df = 3, 12, P = ), 415 (F = 0.58, df = 3, 12, P = 0.642) and 424 (F = 2.38, df = 3, 12, P = ), indicating that they were resistant to deterioration of this trait. Reductions in virulence with increasing number of passages were observed for all other treatments (Fig. 3.5). In line 123, there were no differences in virulence between first, fifth and tenth passages, but virulence after the fifteenth passage was lower than after the first and fifth passages (F = 6.34, df = 3, 12, P = 0.008). In line 125, no differences were observed among the first, fifth, and tenth passages, but efficacy after the fifteenth passage was lower than after the fifth passage (F = 5.70, df = 3, 12, P = ). In inbred line 313, virulence after the tenth and fifteenth passages was significantly lower than after the fifth passage (F = 63

77 5.73, df = 3, 12, P = ). In line 421, virulence was lower after the tenth and fifteenth passages than after the first and fifth passages (F = 7.98, df = 3, 12, P = ). In the sub-cultured wild-type treatment, efficacy after ten passages was lower than after the first and fifth passages (F = 10.93, df = 2, 8, P = ), indicating a degree of trait instability in this line, which was only maintained for five passages. The most pronounced reduction in efficacy with serial culturing was observed in the non-cultured wild-type population; efficacy after the tenth and fifteenth passages was significantly lower than after the first and fifth passages (F = 12.31, df = 3, 12, P = ). The effect of serial culturing on reproductive capacity was also examined and illustrated in figure 3.6. The significant differences in reproductive capacity among treatments were observed after the first passage (F = 2.75, df = 11, 44, P = ). Production of inbred lines 222 and 315 was superior to that of inbred line 415 (Fig. 3.6A). There were no significant differences observed among the other lines, however, it was observed that all the inbred lines, except 415 and the wild-type parents, appeared to have similar potentials for nematode production. Significant differences in reproductive capacity among treatments were observed after the fifth passage (F = 17.22, df = 11, 44, P < ). The highest reproductive capacity was observed in inbred lines 222, 225 and 324, and was significantly greater than the non-cultured population, inbred lines 125, 415, 424 and the subcultured population (Fig. 3.6B). Inbred line 315 had higher reproductive capacity than inbred lines 125, 415 and 424, and the sub-cultured parental line. Inbred line 421 had the third highest reproductive capacity, producing more IJs than inbred lines 125 and 415, and the sub-cultured parental line. 64

78 Figure 3.5. Effects of serial culturing on the virulence (mean ± SE percent efficacy) of 10 homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, and non-cultured and sub-cultured lines of their wild-type parents, against Galleria mellonella larvae. All efficacy trials were carried out at 22 o C, and efficacy was assessed after the first, fifth, tenth and fifteenth passages through G. mellonella (P 0.05). 65

79 Line 123 Line 125 Line 222 Line 225 Line 313 Line 315 Line 324 Line 415 Line 421 Line 424 sub-cultured non-cultured Line 123 Line 125 Line 222 Line 225 Line 313 Line 315 Line 324 Line 415 Line 421 Line 424 non-cultured IJs/cadavers (x 1000) e e d bcd cd abc ab ab a abc a a IJs/cadavers (x 1000) Line 123 Line 125 Line 222 Line 225 Line 313 Line 315 Line 324 Line 415 Line 421 Line 424 sub-cultured non-cultured Line 123 Line 125 Line 222 Line 225 Line 313 Line 315 Line 324 Line 415 Line 421 Line 424 sub-cultured non-cultured IJs/cadaver (x 1000) IJs/cadaver (x 1000) f ef de cde bcd abcd a a abcd ab a abc A ab ab a ab ab a ab b ab ab ab ab B C Treatments D ab bc ab ab Treatments ab ab a d ab bc cd 0 0 Treatments Treatments Figure 3.6. Mean reproductive capacity (± SE) of 10 homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora non-cultured and sub-cultured populations of their wild-type parents. Figures show mean number of IJs per cadaver (x 1000) after the first (A), fifth (B), tenth (C) and fifteenth (D) passage through Galleria mellonella larvae. All assays were run at 22 o C. Bars with the same letter are not significantly different (P 0.05). 66

80 The reproductive capacity of inbred lines 123, 313, and the non-cultured parental line was superior to the sub-cultured parental line and inbred line 415. Inbred lines 125 and 424 had greater reproductive capacity than inbred line 415. There was no difference in the reproductive capacity of the sub-cultured population and inbred line 415. After the tenth passage, reproductive capacity also differed significantly among treatments (F = 27.06, df = 11, 44, P < ). The highest reproductive capacity was shown by inbred lines 313, 324 and 421, and was significantly higher for these 3 lines than for inbred lines 125, 415 and 424, the non-cultured parental population, and the sub-cultured parental line (Fig. 3.6C). Inbred lines 222 and 225 had the next highest level of production which was greater than that of inbred lines 415 and 424, the non-cultured parental population, and the sub-cultured population. The reproductive capacity of inbred lines 123 and 315 was higher than that of the non-cultured parental line, inbred line 415, and the subcultured parental line. The reproductive capacity of inbred lines 125 and 424, and the noncultured parental line was higher than that of inbred line 415 and the sub-cultured parental population. No difference was observed between inbred line 415 and the sub-cultured parental line. Significant differences in reproductive capacity among treatments were also detected after the fifteenth passage (F = 12.95, df = 10, 40, P < ). Reproductive capacity of inbred line 324 was superior to inbred lines 125, 415 and 424, and the non-cultured parental line (Fig. 3.6D). Inbred lines 123, 222, 225, 313, 315 and 421 showed significantly higher reproductive capacity than the non-cultured parental line and inbred line 415. The reproductive capacity of inbred lines 125 and 424 was greater than that of inbred line

81 There was no difference in reproductive capacity between the non-cultured parental line and inbred line 415. No deterioration of reproductive capacity as a result of serial culturing was observed in seven inbred lines: 123 (F = 2.88, df = 3, 12, P = ), 125 (F = 1.05, df = 3, 12, P = ), 225 (F = 1.48, df = 3, 12, P = 0.269), 313 (F = 2.63, df = 3, 12, P = ), 324 (F = 1.00, df = 3, 12, P = ), 421 (F = 2.81, df = 3, 12, P = ) and 424 (F = 1.68, df = 3, 12, P = ) (Fig. 3.7). The reproductive capacity of inbred lines 222 (F = 3.38, df = 3, 12, P = ), 315 (F = 4.98, df = 3, 12, P = ), and 415 (F = 4.23, df = 3, 12, P = ) was higher in the first than the fifteenth passage. The reproductive capacity of the sub-cultured population decreased for each recorded passage (F = 51.28, df = 2, 8, P < ); and, in the non-cultured parental line reproductive capacity was lower after the fifteenth than the first passage (F = 5.31, df = 2, 12, P = ), indicating that this trait was unstable in the wild-type parents and reproductive capacity declined through the serial culturing process. 3.5 Discussion The development of inbred lines of H. bacteriophora proved to be a successful means of maintaining beneficial traits when the nematodes were repeatedly cultured through G. mellonella. Initially, inbred lines were not significantly more virulent or more productive 68

82 Figure 3.7. Mean reproductive capacity (± SE) of 10 homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured lines of the wild-type parents. Figures presented show the mean number of IJs produced (x1000) per Galleria mellonella cadaver. Production is shown for the first, fifth, tenth and fifteenth passages through G. mellonella larvae (P 0.05). All assays were conducted at 22 C. 69

83 than the non-cultured wild-type populations from which they were derived; however a number of inbred lines were able to maintain traits over 15 serial passages through G. mellonella, while the wild-types were not. Those lines that showed no deterioration in the measured traits after serial sub-culture are more desirable as biological control agents and show characteristics that are desirable for commercialization as biological control products. Overall, inbred line 421 was the superior line in both small and large-scale reproductive capacity trials. Inbred lines 324, followed by 421 were the best lines in the shelf-life trials, while line 125 and the sub-cultured parental line showed inferior stability in storage. Based on data obtained from both the virulence and reproductive capacity (after serial sub-culturing) trials, inbred line 324 showed superior characteristics, whereas 415 and the non-cultured wild-type lines had the worst results. Inbred line 123 showed promising efficacy through the virulence trial. Bilgrami et al. (2006) showed that virulence started to decline in experimental H. bacteriophora lines after the tenth passage compared to the control (low-cultured treatment which was four passages in total). However, after 20 passages only 2 of the 5 experimental lines deteriorated in virulence throughout the Steinernema carpocapsae treatments tested in the same trial. Moreover, in the reproductive capacity trial, reduction in this trait occurred in one of the H. bacteriophora samples after just five passages, while after the tenth passage it was observed for all of the experimental lines. In a comparable study, Bai et al. (2005) showed that reduction in efficacy after serial culturing was only observed in the foundation population which was created by mixing IJs obtained from different H. bacteriophora isolates. There was no reduction in virulence after 16 passages for all inbred lines. Reproductive capacity was stable up to the sixteenth passage 70

84 in all inbred lines, contrary to the foundation population where reproductive capacity declined after the eleventh passage. Taking an overview of the data obtained data from all of the trials reported in this chapter, the performance of the inbred lines could be categorised into three major groups; superior, intermediate and inferior. Inbred line 324 may be regarded as an elite line, showing superior stability in storage, while also displaying high virulence and reproductive capacity. Inbred line 123 could also be placed in this category, since it demonstrated high reproductive capacity in small-scale trials, displayed excellent stability in storage and maintained virulence through the fifteenth passage. Three inbred lines could be classified as intermediate, including 222, 225 and 421. Inbred line 222 maintained high virulence through the serial culturing process, as well as high reproductive capacity in the small-scale trials and the serial culturing trials. However, these traits were not maintained in the large-scale production trials and it showed poor storage characteristics. While inbred line 225 showed no reduction in efficacy through serial culturing, it was not as virulent as other lines. No decrease was recorded for its reproductive capacity. Inbred line 415 may be considered as inferior to the rest of the lines tested. This line exhibited very poor productivity in both small and serial culturing trials. Also, it showed lower virulence through all of the assessments in the serial culturing trial, and viability was significantly lower than for other lines after 80 days in storage. This variation among traits could be expected within the inbred lines, and such variation might be anticipated in a wild-type population. However, the results obtained 71

85 clearly showed that lines showing a higher level of performance and stability of desired traits can be created (Bai et al., 2005). Of significance was the loss of desirable traits by the non-cultured parental line after repeated passages through a susceptible host. Both efficacy and reproductive capacity declined, and become more significant with increasing passages. There was a positive correlation between the number of serial passages and observations of differences between treatments. This confirmed the hypothesis that the inbred lines will generally remain stable through serial culturing, whereas wild-type populations are more prone to a loss of these beneficial traits with or without repeated culturing. This confirmed our hypothesis that creating homozygous inbred lines could be a solution against deterioration of beneficial traits in economically-important EPNs. This study demonstrated that inbred lines are more stable through serial culturing than wild-types. Overall, inbred lines 123 and 324 displayed excellent traits and had a longer storage life than the current commercial lines. As such, it is recommended that these elite lines be taken into commercial production. Future studies should focus on use of the inbred line technique to improve additional traits such as: field performance, host seeking ability, performance at lower temperatures, desiccation tolerance and adaptability to in vitro production techniques. 72

86 CHAPTER FOUR Efficacy of homozygous inbred lines of Heterorhabditis bacteriophora against the greater wax moth, Galleria mellonella, and cabbage maggot, Delia radicum, under relatively cold conditions 4.1 Abstract Serial culturing can lead to deterioration in numerous beneficial traits of entomopathogenic nematodes (EPN) including virulence, stability in storage, longevity, heat tolerance, etc. This problem can be eliminated or reduced by creating homozygous inbred lines. Efficacy at low temperatures is a concern when utilizing EPNs in Canada for pest management. To address this, 10 homozygous lines were created from a local Heterorhabditis bacteriophora strain by using the inbred line technique and their efficacy was evaluated in two trials against larvae of the greater wax moth, G. mellonella, and the cabbage maggot, Delia radicum, under low temperature conditions. To assess the impact of serial culturing on nematode performance, two samples of wild-type parents ( non-cultured, the original parent population, and sub-cultured, a serial cultured population) were also included in these experiments. The first trial evaluated efficacy against 7 th instar G. mellonella under five constant temperature regimes, i.e. 8, 12, 16, 20 and 24 o C. No differences in efficacy were detected among EPN treatments at 8 and 12 o C. At 16 o C, eight inbred lines and the non-cultured parents demonstrated efficacy against G. mellonella. At the warmest temperatures, all EPN treatments were effective. In the subsequent experiment, the efficacy of the EPN lines against D. radicum was evaluated at 16 o C, using two test 73

87 concentrations; two commercial strains of H. bacteriophora were also included. All treatments except inbred line 125 caused higher levels of infection than the control. The majority of the inbred lines and the non-cultured wild-type population exhibited superior efficacy compared to the sub-cultured parent population, and both commercial strains. Key words: Serial culturing, cold tolerance, entomopathogenic nematodes, virulence 4.2 Introduction Performance of imported entomopathogenic nematodes (EPNs) in Ontario s cool spring and autumn conditions is a key factor for growers considering using them for pest control. Poor field efficacy has been reported for an imported strain of H. bacteriophora, perhaps due to poor adaptation of this strain to cool soil conditions (Kaya, et al., 2006). Low survival levels and inaccurate application times contribute to the lack of control observed under such conditions (Georgis et al., 2006). Efficacy, reproduction and establishment of different EPN species can occur over a wide range of temperatures, e.g., infection by Steinernema riobrave and H. bacteriophora can occur between 10 to 39 o C and 10 to 32 o C, respectively (Grewal et al., 1994). Robust local strains of EPNs, which are highly efficacious against native insect pests, may be better adapted to the prevailing environmental conditions. Development of such strains as biopesticides could enable insect pests in cooler soils to be more effectively controlled. The creation of inbred lines can enhance genetic stability and permit the production of an elite line in which desirable characteristics are inherently more stable than the parental 74

88 wild-type (Bai et al., 2005). Selection of lines which perform well at lower temperatures could increase the likelihood of successful pest control during the spring and fall in more temperate climates. In addition, locally-based EPN products may help to reduce some of the costs associated with the use of EPNs in pest management, by requiring reduced concentrations and/or numbers of applications throughout a season (Shields, et al., 2009). In Ontario, many of the dominant insect species overwinter in the soil, as last instar larvae: e.g., European chafer, Rhizotrogus majalis (Coleoptera: Scarabaeidae) and Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae); or as pupae, e.g., imported cabbageworm, Artogeia rapae (Lepidoptera: Pieridae), and the cabbage maggot, Delia radicum (Diptera: Anthomyiidae). (Chaput, 1999; Charbonneau, 2009). In the spring, there is a window of opportunity to utilise EPNs that can tolerate cool conditions to target soil-dwelling stages of important pest insects that overwinter in the soil before they emerge as adults. Pest management actions targeted at overwintering populations may also limit subsequent population growth and economic damage (Brunner et al., 1993). Logically, a locally adapted EPN with a high level of virulence is the best candidate for this task. In this study, an array of homozygous inbred lines was created with emphasis on selection for enhanced cold tolerance and virulence. Use of the inbred line process could maintain such beneficial characteristics and protect lines against trait deterioration through serial culturing. In this study, inbred lines were compared for trait stability and efficacy relative to the wild-type parents against two study organisms; G. mellonella (Lepidoptera: Pyralidae), and D. radicum. 75

89 Galleria mellonella is a factitious host for EPN research and production, owing to its high level of susceptibility to various EPN species, and its ready availability, while D. radicum is one of the most important insect pests of Brassica crops in Canada (Dixon, 2015). There are three generations of D. radicum per year in Ontario (OMAFRA, 2009). The first generation, which is the most damaging, occurs from mid-may to early June, the second from late June to mid-july, and the third from late August to early September (OMAFRA, 2009), which coincide with average monthly temperatures of 15.1, 21.5 and 19.3 o C, respectively (Fig.4.1). All life stages of D. radicum, with the exception of adults, occur on or in the soil (thus making them a good target for control with EPNs), and the larvae can cause extensive root and tuber damage leading to economic losses (Charbonneau, 2009). Therefore, our hypothesis was that inbred line techniques can be used to increase efficacy characteristics of EPNs at cool temperatures and thus improve opportunities for their successful use under the climatic conditions of southern Ontario. 4.3 Materials and methods Cool temperature assessment trial against Galleria mellonella The virulence of 10 homozygous inbred lines derived from a local strain of H. bacteriophora (see Chapter 2) and their wild-type parents was assessed at a range of temperatures (8, 12, 16, 20 and 24 o C) in an experiment conducted at the School of Environmental Sciences, University of Guelph, Guelph, ON. The experiment included 13 treatments: 10 inbred lines, 2 parental lines and a control. The control treatments at each temperature received only distilled water. 76

90 Figure 4.1. The average monthly temperature ( o C) in Toronto, Ontario, based on climate data gathered from 1995 to 2014 ( 2015). The three generations of cabbage maggot, Delia radicum, that occur in southern Ontario are represented by the bars above the graph. 77

91 To determine effects of serial culturing on EPN traits, two parental line treatments based on the original wild-type population, with and without serial culturing, were included. These were specified as non-cultured (the initial population after soil extraction) and subcultured (the original population after 15 passages through G. mellonella). To remove infective juvenile (IJ) age as a factor in this experiment, G. mellonella larvae were inoculated with all of the inbred lines and wild-type parents 21 days before the trial began. Infective juveniles of the inbred lines and wild-type parents were previously stored in flasks with the concentration of 500 IJs/ml at 13 o C prior to the inoculation of the wax moth larvae. The IJs obtained from the G. mellonella larvae were stored in aerated containers at 13 o C for approximately seven days, until the start of the experiment. Each experimental unit consisted of a medium sized Petri dish (100 x 15 mm) lined with a single layer of Grade 202 filter paper (Reeve Angel, Boreal Science, St. Catharines, ON) and containing ten healthy 7 th instar G. mellonella (Vanderhorst Wholesaler Inc., St. Marys, OH). Prior to placement in the dish, each larva was inoculated by immersion in a suspension of 7-10 day old nematodes (1000 IJs/ml) for 30 sec. Petri dish lids were secured in place with Parafilm and were arranged in a randomized manner on five laboratory trays. Each tray was assigned to one of five incubators, the fridge model for 8 o C (Model R05REC, WC Woods Co., Guelph, Ontario), was different with other temperatures (Model E7H, Controlled Environments Ltd., Winnipeg, Manitoba); each being set at a different temperature. The dishes were arranged in a randomized complete block design, with 5 replications per temperature * treatment. There were 65 Petri dishes on each tray, and trays were positioned on the middle rack where there is better air circulation and conditions less prone to fluctuation. 78

92 Each incubator was calibrated for each experimental temperature using an accurate digital thermometer (Model CON4040, Fisher Scientific, Ottawa, Ontario) installed in each incubator and checked for seven days at 8 h intervals prior to the start of the trial. A thermometer/hygrometer (Model CON4096, Fisher Scientific Canada, Ottawa, Ontario) was placed inside each incubator to record internal environmental conditions during the trial. The lights were kept off throughout the trial to avoid any side effects of light on nematode performance. All dishes were inspected at 48 h intervals and the number of infected larvae recorded. To minimize effects of temperature change during the examination, only one tray of Petri dishes was removed from the incubator at a time for counting, and was returned before the next tray was selected. This way, each dish spent less than 10 min at room temperature per observation period. Larvae were considered to have been successfully infected when the cadavers had a pink/red coloration. The number of non-infected larvae that were either alive or dead due to putative other causes (not showing typical signs of EPN infection e,g., black or beige coloration), were also recorded. The experiment was monitored for two weeks to account for (potentially) prolonged penetration time, bacterial development time, and time for the cadavers to change colour, particularly under the low temperature regimes (Griffin and Downes, 1994) Efficacy of inbred lines against Delia radicum under cool conditions Delia radicum pupae that had completed diapause were provided by the London Research & Development Centre, Agriculture and Agri-Food Canada, London, ON. Delia 79

93 radicum were reared for the trials using methods described by Whistlecraft et al. (1985) and Harris and Svec (1966). In brief, open Petri dishes containing pupae were placed inside a rearing cage and observed daily for adult emergence. Moisture, which is necessary for adult emergence (Delahaut, 2003) was provided by inserting a wet rolled filter paper or a dental wick, through a small hole in the lid of a water container (Fig. 4.2A). After adults emerged, several oviposition devices were introduced in to the rearing cage (Fig. 4.2B). Each oviposition device consisted of a 9 cm Petri dish lid or base (i.e. water reservoir) and a 15 cm Petri dish with a 1 cm hole in the centre was glued on to the 9 cm dish. A rolled tissue or a dental wick was placed in the hole and inserted into the water. The wick was covered with a layer of filter paper, and was kept continually moist by distilled water transferred up from the reservoir. Five hundred gm of organic rutabaga (as an oviposition stimulant), were placed in the center of the dish, and were covered by an inverted miniature clay pot. The gap between the clay pot and the larger petri-dish was filled with activated charcoal to provide a substrate for egg deposition. After seven days, eggs were recovered from the charcoal by sieving through two mesh screens (3 mm and 150 µm) (Fig. 4.2C). Subsequently, eggs were introduced to D. radicum rearing containers which contained rutabaga pieces pushed partially into moistened silty soil. First instar D. radicum fed on the rutabaga and after three weeks, third instars that were >6mm in length were collected for use in the experiment (Fig. 4.2D). Ten larvae were used in each experimental unit; when removed from the infested rutabaga they were held in small plastic containers prior to being introduced into the experimental pots. 80

94 A B C D Figure 4.2. Rearing container for Delia radicum maggots on rutabaga media (A) Top view of D. radicum rearing cage, containing water containers with rolled filter paper wicks to provide the desired moisture. (B) Oviposition device, consisting of organic rutabaga pieces and activated charcoal placed on a petri dish and covered with inverted clay pots. (C) Delia radicum eggs were collected on a fine screen. (D) Delia radicum maggots were fed on pieces of organic rutabaga. Photographs: S. Sharififar. 81

95 In this trial each experimental unit consisted of a 15 cm diameter plastic pot containing a single hybrid between Chinese broccoli and Guy-Lon plant, Brassica oleracea var. alboglabra (Stokes Seeds Limited. Thorold, Ontario), with 4-6 fresh green leaves grown in a medium of Promix soil (Premier Tech Horticulture, Rivière-du-Loup, Québec). A small quantity of potting mix was removed from each pot to create a shallow trench (approx. 2 cm deep) around each broccoli seedling; 10 maggots (Fig. 4.3A) were placed in the trench (Fig. 4.3B) and were then covered by the potting mix. Fifty ml of nematode suspension was applied to each pot. A total of 15 treatments were included in this experiment: 10 inbred lines, two parental lines (with and without serial culturing), two commercially available strains as Commercial 1 and commercial 2 as well as a control (distilled water only). Two IJ concentrations were used for each EPN treatment (4,000 and 40,000 IJs/pot), for a total of 29 experimental treatments. Pots were arranged in a randomized complete block design, and each treatment was replicated 5 times. After introduction of the EPNs, each pot was covered with a fine mesh bag to capture adult D. radicum when they emerged. All pots were held inside a growth chamber at 16 o C with a 14:10 hour light/dark cycle (Fig. 4.3C). The soil moisture in all pots was maintained by topwatering with 100 ml per pot at 96 hour intervals. All mesh bags were adjusted weekly basis as the plants grew. Adults that emerged were removed and counted every day for 4 weeks. After 5 weeks, plants were removed from the soil, roots were washed and the root system was inspected for maggot-feeding damage (Fig. 4.3D). Damage was rated on a 1 to 5 scale based on the extent of root growth and was categorized according to the size of the root system, with the greatest development scored as 5 or strong, and the smallest scored as 1 or weak. Three other intermediate ranges were classified by root size as 2 = weak-medium, 3 = 82

96 A B C D Figure 4.3. (A) Ten first instar Delia radicum ready to be introduced to an experimental pot. (B) Artificial infestation of the broccoli plant; cabbage maggots were placed in the trench prepared around the plant and covered with potting mix. (C) Experimental pots in the incubator (16 o C, 14:10 hours day/night photoperiod). (D) Broccoli root system assessed for maggot feeding damage and growth. Photographs: S. Sharififar. medium, and 4 = medium-strong. 83

97 4.3.3 Statistical analyses All statistical analyses were conducted using SAS 9.3 software. Data were analyzed using ANOVA at α = 0.05, followed by Tukey s test for means separations. For the Galleria trial, ANOVA was applied to first detect significant treatment effects. When a significant F- value was detected, Tukey's test was used to further elucidate differences among the treatments; percentage data (percent infection) were transformed using arcsine or square root transformation prior to analysis. Residuals were plotted against any time variables present to validate the model and the independence of any differences detected. For both cabbage maggot trials, analyses of root damage and maggot control were conducted using a generalized linear mixed model variance analysis (GLIMMIX) procedure. Inbred lines were fixed effects and replications were random effects. A multiple means comparison on the model scale was performed at P = 0.05 using Tukey's test. Estimates and standard error were converted to the data scale using the ilink option. Plots of studentized residuals were used to confirm the analysis assumptions. The estimates are the means on the analysis scale (logit) and the means are these estimates backtransformed to the original scale. Also, for cabbage maggot control, as a qualitative trial, a beta distribution, with logit link was fit using the Laplace approximation. Additionally, a slight adjustment to the 0 and 1 values; a beta distribution required 0<y<1 so this brings the 1 values in ever so slightly so they are retained in the analysis. Moreover, to investigate any interaction between lines and density, low and high, the obtained data was analysed as a randomized complete block with a factorial design. 84

98 4.4 Results Cool temperature assessment trial against Galleria mellonella For trials run at 8 o C, ANOVA indicated significant differences in mortality among lines or the control, but no differences were detected among the means by the subsequent Tukey s test (F = 2.25, df = 12, P = ) (Fig. 4.4). At 12 o C, there were no significant differences in mortality among lines or the control (F = 1.88, df = 12, P = ) (Fig. 4.5). At 16 o C, significant differences in efficacy were found among lines (F = 5.99, df = 12, P< ) (Fig. 4.6). All lines, except inbred lines 222, 421 and the sub-cultured parental line, caused significantly more mortality than the control. In addition, five of the inbred lines (123, 125, 225, 313, and 324), caused significantly higher rates of infection than inbred line 421. At 20 o C, inbred line 222 exhibited higher infectivity than line 225, but all treatments caused higher mortality than the control (F = 9.99, df = 12, P < ) (Fig. 4.7). At 24 o C, higher infection levels were observed in all treatments relative to the control (F = 12.93, df = 12, P < ) (Fig. 4.8). Inbred line 222 caused significantly higher rates of infection than lines 415, 424, and the sub-cultured parental population. Inbred line 421 also induced a higher rate of infection compared to inbred line 415 and the sub-cultured population Efficacy of inbred lines against Delia radicum under cool conditions All nematode treatments, with the exception of both rates of inbred line 125, caused higher levels of D. radicum mortality (as evidenced by reduced adult emergence rates) than the control (F = 10.89, df = 28, P < ) (Fig. 4.9). The most effective lines for cabbage maggot control were inbred lines 123, 222, 225, 315, 324, 415, 421, 424, the non-cultured 85

99 Figure 4.4. Mean percent (± SE) mortality of Galleria mellonella larvae after treatment with homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, at 8 o C. The sub-cultured population underwent 15 serial passages through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Bars with the same letter are not significantly different (P 0.05). 86

100 Figure 4.5. Mean percent (± SE) mortality of Galleria mellonella larvae after treatment with homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, at 12 o C. The sub-cultured population underwent 15 serial passages through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Bars with the same letter are not significantly different (P 0.05). 87

101 Figure 4.6. Mean percent (± SE) mortality of Galleria mellonella larvae after treatment with homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, at 16 o C. The sub-cultured population underwent 15 serial passages through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Bars with the same letter are not significantly different (P 0.05). 88

102 Figure 4.7. Mean percent (± SE) mortality of Galleria mellonella larvae after treatment with homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, at 20 o C. The sub-cultured population underwent 15 serial passages through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Bars with the same letter are not significantly different (P 0.05). 89

103 Figure 4.8. Mean percent (± SE) mortality of Galleria mellonella larvae after treatment with homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, at 24 o C. The sub-cultured population underwent 15 serial passages through G. mellonella, while the non-cultured population was the original population collected after soil extraction. Bars with the same letter are not significantly different (P 0.05). 90

104 Figure 4.9. Mean percent (± SE) mortality of cabbage maggot, Delia radicum, larvae after treatment with homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, and two commercial strains at 16 o C. Each treatment was applied at two rates low (L, 4000 IJs/pot) and high (H, 40,000 IJs/pot). The control treatment received 50 ml. of distilled water only. Bars with the same letter are not significantly different (P 0.05). 91

105 parental population at both rates, and inbred line 313, commercial lines 1 and 2 at the high rate. The low rates of inbred line 313, the sub-cultured parental line and commercial lines 1 and 2, as well as both rates of inbred line 125 caused the lowest levels of mortality. Nematode concentration (F = 20.22, df = 1, 116, P < ) and the interaction between treatment and concentration (F = 2.04, df = 14, 116, P = ) had a significant influence on root maggot mortality. The mortality among treatments at the high test concentration was approximately five percent greater than at the low concentration, although the difference was greater for several lines (e.g., 313 and commercial 1 with 20 and 16% higher mortality at the high vs low concentration, respectively) Root damage assessment results There was a significant difference in root system damage among treatments (F = 5.88, df = 28, 112, P < ). Root quality was best following treatment with the high concentration of inbred line 324, and was better than both concentrations of the sub-cultured parental line and inbred line 125, and the low concentrations of inbred lines 123, 225, 313, 421 and 424, both commercial lines and the control (Fig. 4.10). 4.5 Discussion Access to elite nematode strains, regardless of the culture method, is essential to advance nematode production on a commercial scale. Such strains provide advantages for both the producer and consumer: higher reproductive capacity should reduce production costs and higher virulence may allow application rates to be reduced as well (Shapiro-Ilan et al., 2012). 92

106 Figure Mean values of the qualitative point score (± SE) assigned to broccoli roots after five weeks. The best root condition was assigned a value of 5, while the worst (damaged) roots received one point. Damage assessments made after infestation of broccoli plants with cabbage maggot, Delia radicum, larvae, and application of homozygous inbred lines derived from a local strain of Heterorhabditis bacteriophora, non-cultured and sub-cultured populations of their wild-type parents, and two commercial strains, at 16 o C. Treated pots were examined after 5 weeks. Each treatment was applied at two rates: low (L, 4000 IJs/pot) and high (H, 40,000 IJs/pot). The control treatment received 50 ml. of distilled water only. Bars with the same letter are not significantly different (P 0.05). 93