Control of Burrowing Nematodes

Control of Burrowing Nematodes.

Biological suppression of root-lesion nematodes in grain-growing soils

Root-lesion nematodes (RLNs) are found on 75% of grain farms in southern Queensland (QLD) and northern New South Wales (NSW) and are significant pests. This project confirmed that biological suppression of RLNs occurs in soils, examined what organisms are involved and how growers might enhance suppressiveness of soils. Field trials, and glasshouse and laboratory bioassays of soils from fields with contrasting management practices, showed suppressiveness is favoured with less tillage, more stubble and continuous intensive cropping, particularly in the top 15cm of soil. Through extensive surveys key organisms, Pasteuria bacteria, nematode-trapping fungi and predatory nematodes were isolated and identified as being present.

Resistance to root-lesion Nematodes for development of molecular markers

This is a sub-project of the Australian Wheat and Barley Molecular Marker Program funded by GRDC and led by Drs Diane Mather and Ken Chalmers of University of Adelaide. In this sub-project we will supply phenotypic data on resistance to two species of root-lesion nematodes (Pratylenchus thornei and P. neglectus) on several populations of wheat doubled haploids. We will also supply existing genotypic data on one doubled haploid population. We will also test one population of doubled haploids (CPI133872/Janz) a second time for resistance to P. thornei and P. neglectus and supply this information to University of Adelaide for the development of molecular markers for use by wheat breeders in selecting for resistance to root-lesion nematodes.

Improving oncolytic adenoviral therapies for gastrointestinal cancers and tumor initiating cells

Although the treatment of most cancers has improved steadily, only few metastatic solid tumors can be cured. Despite responses, refractory clones often emerge and the disease becomes refractory to available treatment modalities. Furthermore, resistance factors are shared between different treatment regimens and therefore loss of response typically occurs rapidly, and there is a tendency for cross-resistance between agents. Therefore, new agents with novel mechanisms of action and lacking cross-resistance to currently available approaches are needed. Modified oncolytic adenoviruses, featuring cancer-celective cell lysis and spread, constitute an interesting drug platform towards the goals of tumor specificity and the implementation of potent multimodal treatment regimens. In this work, we demonstrate the applicability of capsid-modified, transcriptionally targeted oncolytic adenoviruses in targeting gastric, pancreatic and breast cancer. A variety of capsid modified adenoviruses were tested for transductional specificity first in gastric and pancreatic cancer cells and patient tissues and then in mice. Then, oncolytic viruses featuring the same capsid modifications were tested to confirm that successful transductional targeting translates into enhanced oncolytic potential. Capsid modified oncolytic viruses also prolonged the survival of tumor bearing orthotopic models of gastric and pancreatic cancer. Taken together, oncolytic adenoviral gene therapy could be a potent drug for gastric and pancreatic cancer, and its specificity, potency and safety can be modulated by means of capsid modification. We also characterized a new intraperitoneal virus delivery method in benefit for the persistence of gene delivery to intraperitoneal gastric and pancreatic cancer tumors. With a silica implant a steady and sustained virus release to the vicinity of the tumor improved the survival of the orthotopic tumor bearing mice. Furthermore, silica gel-based virus delivery lowered the toxicity mediating proimflammatory cytokine response and production of total and anti-adenovirus neutralizing antibodies (NAbs). On the other hand, silica shielded the virus against pre-excisting NAbs, resulting in a more favourable biodistribution in the preimmunized mice. The silica implant might therefore be of interest in treating intraperitoneally disseminated disease. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in cancer relapse and the formation of metastasis. Therefore, we examined if transcriptionally modified oncolytic adenoviruses are able to kill these cells. Complete eradication of CD44+CD24-/low putative breast cancer stem cells was seen in vitro, and significant antitumor activity was detected in CD44+CD24-/low –derived tumor bearing mice. Thus, genetically engineered oncolytic adenoviruses have potential in destroying cancer initiating cells, which may have relevance for the elimination of cancer stem cells in humans.

Cropping options to limit root-lesion nematodes

Australia’s northern grain-producing region is unique in that the root-lesion nematode (RLN), Pratylenchus thornei predominates. P. neglectus is also present. RLN cause substantial yield losses, particularly in wheat, but they reproduce on numerous summer and winter crops. Each nematode species prefers different crops and varieties. This project provides growers with a range of integrated management strategies to limit RLN (i.e. identify the problem, protect uninfested fields, rotate with resistant crops to keep populations low and choose tolerant crops to maximise yields). It also provides new information about soil-borne zoosporic fungi in the region.

Synthetic Evaluation Project - Nematodes

The project tests synthetic hexaploid wheats for resistance to root-lesion nematodes.

Hybridisation of Australian chickpea cultivars with wild Cicer spp. increases resistance to root-lesion nematodes (Pratylenchus thornei and P. neglectus).

Australian and international chickpea (Cicer arietinum) cultivars and germplasm accessions, and wild annual Cicer spp. in the primary and secondary gene pools, were assessed in glasshouse experiments for levels of resistance to the root-lesion nematodes Pratylenchus thornei and P. neglectus. Lines were grown in replicated experiments in pasteurised soil inoculated with a pure culture of either P. thornei or P. neglectus and the population density of the nematodes in the soil plus roots after 16 weeks growth was used as a measure of resistance. Combined statistical analyses of experiments (nine for P. thornei and four for P. neglectus) were conducted and genotypes were assessed using best linear unbiased predictions. Australian and international chickpea cultivars possessed a similar range of susceptibilities through to partial resistance. Wild relatives from both the primary (C. reticulatum and C. echinospermum) and secondary (C. bijugum) gene pools of chickpea were generally more resistant than commercial chickpea cultivars to either P. thornei or P. neglectus or both. Wild relatives of chickpea have probably evolved to have resistance to endemic root-lesion nematodes whereas modern chickpea cultivars constitute a narrower gene pool with respect to nematode resistance. Resistant accessions of C. reticulatum and C. echinospermum were crossed and topcrossed with desi chickpea cultivars and resistant F(4) lines were obtained. Development of commercial cultivars with the high levels of resistance to P. thornei and P. neglectus in these hybrids will be most valuable for areas of the Australian grain region and other parts of the world where alternating chickpea and wheat crops are the preferred rotation.

Inheritance of resistance to root-lesion nematodes (Pratylenchus thornei and P. neglectus) in five doubled-haploid populations of wheat

Nematode species Pratylenchus thornei and P. neglectus are the two most important root-lesion nematodes affecting wheat (Triticum aestivum L.) and other grain crops in Australia. For practical plant breeding, it will be valuable to know the mode of inheritance of resistance and whether the same set of genes confer resistance to both species. We evaluated reactions to P. thornei and P. neglectus of glasshouse-inoculated plants of five doubled-haploid populations derived from five resistant synthetic hexpaloid wheat lines, each crossed to the susceptible Australian wheat cultivar Janz. For each cross we determined genetic variance, heritability and minimum number of effective resistance genes for each nematode species. Distributions of nematode numbers for both species were continuous for all doubled-haploid populations. Heritabilities were high and the resistances were controlled by 4-7 genes. There was no genetic correlation between resistance to P. thornei and to P. neglectus in four of the populations and a significant but low correlation in one. Therefore, resistances to P. thornei and to P. neglectus are probably inherited quantitatively and independently in four of these synthetic hexaploid wheat populations, with the possibility of at least one genetic factor contributing to resistance to both species in one of the populations. Parents with the greatest level of resistance will be the best to use as donor parents to adapted cultivars, and selection of resistance to both species in early generations will be optimal to carry resistance through successive cycles of inbreeding to produce resistant cultivars for release.

Biological Control of Oilseed Rape Pests with Entomopathogenic Nematodes

Biological control techniques attract increasing attention as one of the sustainable alternatives to pesticide use in integrated pest management programs. In order to develop sustainable pest management methods for arable crops based on entomopathogenic nematodes (EPN), their efficacy and persistence needed to be investigated, and an economically feasible delivery system had to be developed. In this study, first a survey of entomopathogens was conducted, and a system approach was tested, using the oilseed Brassica (OSB) growing system (OSB, spring wheat, and red clover) as a model. The system approach aimed at determining the potential of Steinernema feltiae (Filipjev) for the control of OSB pests, developing OSB rotation schemes that support EPN persistence, and investigating the impact of the selected biotic and abiotic factors on efficacy and persistence of EPN. This study employed abductive logic (which employs constant interplay between the theory and empirical observation), quantitative methods, and a case study on OSB. Laboratory and field experiments were carried out, and two types of pathogen surveys. A horizontal survey included OSB fields across Estonia, Germany, Poland, Sweden and the UK, while a vertical survey included sampling from two sets of differently managed experimental fields during three years. A new approach was introduced for measuring occurrence, where the prevalence and relative intensity of entomopathogens, biotic agents, and unidentified insect antagonists were determined. The effect of dose, timing, and the application method on S. feltiae in the control of pests in OSB, and the potential of a controlled release delivery system (CRS) were evaluated in the field. Studies on the impact of selected biotic and abiotc factors (Brassica plant, bait insects, developmental stages of Meligethes aeneus Fab., Isaria fumosorosea Wize (Ifr), and organic and synthetic fertilizers) on the efficacy of S. feltiae were conducted in the laboratory. Persistence of S. feltiae in the OSB growing system, and the effect of dose, timing, and the application method, was assessed in the field as part of the efficacy experiments. The impact of selected biotic and abiotic factors on S. feltiae persistence was assessed in laboratory experiments. The pathogen survey showed that the occurrence of entomopathogens is low in the OSB growing system, and that a management system causing less disturbance (ICM) to the soil increases the relative intensity of insect parasitic nematodes and other insect antagonists. A longer study period is required to show any possible impact of ICM on the relative intensity of entomopathogenic fungi, or on the prevalence of entomopathogens. Two different measures of the occurrence yielded different results: the relative intensity revealed the difference between the two different crop management methods, while prevalence did not. The highest efficacy of S. feltiae was achieved by using a low dose and targeting all stages of M. aeneus. When only the larval stage was targeted, the application method and dose had no significant effect. The CRS decreased the pest abundance significantly more than the surface application method. S. feltiae persisted in the OSB fields in Finland for several months, but did not survive the winter. The strain survived for 7 months when it was applied in autumn in Germany, but its populations declined rapidly after winter. The examined biotic and abiotic factors had variable impacts on S. feltiae efficacy and persistence. The two measures, prevalence and relative intensity of entomopathogens, gave valuable information for their use in biocontrol programs. The recommended biocontrol strategy for OSB growing in Finland is inundation and seasonal inoculation of EPN. The impact of some biotic and abiotic factors on S. feltiae efficacy and persistence is significant, and can be used to improve the efficacy of EPN. The CRS is a novel alternative for EPN application, and should also be considered for use on other crops. Keywords: Biological control, inundation, inoculation, conservation, formulation, slow release method, crop rotation, Entomopathogenic nematodes, Steinernema feltiae, oilseed rape pests, Meligethes aeneus, Phyllotreta spp., occurrence, prevalence, intensity, efficacy, persistence, field, Isaria fumosorosea, biotic factors, abiotic factors, interaction, impact, insect stages, integrated crop management, standard (conventional) crop management

Cereal cultivars can be ranked consistently for resistance to root-lesion nematodes (Pratylenchus thornei & P. neglectus) using diverse procedures

The root-lesion nematodes (RLN) Pratylenchus thornei and P. neglectus are widely distributed in Australian grain producing regions and can reduce the yield of intolerant wheat cultivars by up to 65 , costing the industry ~123 M AUD/year. Consequently, researchers in the northern, southern and western regions have independently developed procedures to evaluate the resistance of cereal cultivars to RLN. To compare results, each of the three laboratories phenotyped a set of 26 and 36 cereal cultivars for relative resistance/susceptibility to P. thornei and P. neglectus respectively. The northern and southern regions also investigated the effects of planting time and experiment duration on RLN reproduction and cultivar ranking. Results show the genetic correlation between cultivars tested using the northern and southern procedures evaluating P. thornei resistance was 0.93. Genetic correlations between experiments using the same procedure, but with different planting times, were 0.99 for both northern and southern procedures. The genetic correlation between cultivars tested using the northern, southern and western procedures evaluating P. neglectus resistance ranged from 0.71 to 0.95. Genetic correlations between experiments using the same procedure but with different planting times ranged from 0.91 to 0.99. This study established that, even though experiments were conducted in different geographic locations and with different trial management practices, the diverse nematode resistance screening procedures ranked cultivars similarly. Consequently, RLN resistance data can be pooled across regions to provide national consensus ratings of cultivars.

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

Novel matrix metalloproteinases in intestinal inflammation and in cancer of the gastrointestinal tract

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.

Gastrointestinal complications after kidney transplantation

The aim of the study was to evaluate gastrointestinal (GI) complications after kidney transplantation in the Finnish population. The adult patients included underwent kidney transplantation at Helsinki University Central Hospital in 1990-2000. Data on GI complications were collected from the Finnish Kidney Transplantation Registry, patient records and from questionnaires sent to patients. Helicobacter pylori IgG and IgA antibodies were measured from 500 patients before kidney transplantation and after a median 6.8-year follow up. Oesophagogastroduodenoscopy with biopsies was performed on 46 kidney transplantation patients suffering from gastroduodenal symptoms and 43 dyspeptic controls for studies of gastroduodenal cytomegalovirus (CMV) infection. Gallbladder ultrasound was performed on 304 patients after a median of 7.4 years post transplantation. Data from these 304 patients were also collected on serum lipids, body mass index and the use of statin medication. Severe GI complications occurred in 147 (10%) of 1515 kidney transplantations, 6% of them fatal after a median of 0.93 years. 51% of the complications occurred during the first post transplantation year, with highest incidence in gastroduodenal ulcers and complications of the colon. Patients with GI complications were older and had more delayed graft function and patients with polycystic kidney disease had more GI complications than the other patients. H.pylori seropositivity rate was 31% and this had no influence on graft or patient survival. 29% of the H.pylori seropositive patients seroreverted without eradication therapy. 74% of kidney transplantation patients had CMV specific matrix protein pp65 or delayed early protein p52 positive findings in the gastroduodenal mucosa, and 53% of the pp65 or p52 positive patients had gastroduodenal erosions without H.pylori findings. After the transplantation 165 (11%) patients developed gallstones. A biliary complication including 1 fatal cholecystitis developed in 15% of the patients with gallstones. 13 (0.9%) patients had pancreatitis. Colon perforations, 31% of them fatal, occurred in 16 (1%) patients. 13 (0.9%) developed a GI malignancy during the follow up. 2 H.pylori seropositive patients developed gastroduodenal malignancies during the follow up. In conclusion, severe GI complications usually occur early after kidney transplantation. Colon perforations are especially serious in kidney transplantation patients and colon diverticulosis and gallstones should be screened and treated before transplantation. When found, H.pylori infection should also be treated in these patients.

Cow’s milk related gastrointestinal symptoms in adults

The purpose of this study was to evaluate subjective food-related gastrointestinal symptoms and their relation to cow’s milk by determining the genotype of adult-type hypolactasia, measuring antibodies against milk protein, and screening the most common cause for secondary hypolactasia, namely coeliac disease. The whole study group comprised 1900 adults who gave a blood sample for the study when they attended a health care centre laboratory for various reasons. Of these 1885 (99%) completed a questionnaire on food-related gastrointestinal symptoms. Study No. I evaluated the prevalence of adult-type hypolactasia and its correlation to self-reported milk induced gastrointestinal symptoms. The testing for hypolactasia was done by determination of the C/T-13910 genotypes of the study subjects. The results show that patients with the C/C-13910 genotype associated with adult type hypolactasia consume less milk than those with C/T-13910 and T/T-13910 genotypes. Study No. II evaluated the prevalence and clinical characteristics of undiagnosed coeliac disease in the whole study population with transglutaminase and endomysium antibodies and their correlation with gastrointestinal symptoms. The prevalence of coeliac disease was 2 %, which is surprisingly high. Serum transglutaminase and endomysium antibodies are valuable tools for recognising an undiagnosed coeliac disease in outpatient clinics. In the study No. III the evaluation of milk protein IgE related hypersensitivity was carried out by stratifying all 756 study subjects with milk related problems and randomly choosing 100 age and sex matched controls with no such symptoms from the rest of the original study group. In the study No. IV 400 serum samples were randomly selected for analyzing milk protein related IgA and IgG antibodies and their correlation to milk related GI-symptoms. The measurement of milk protein IgA, IgE or IgG (studies No. III and IV) did not correlate clearly to milk induced symptoms and gave no clinically significant information; hence their measurement is not encouraged in outpatient clinics. In conclusion, adult type hypolactasia is often considered the reason for gastrointestinal symptoms in adults and determination of the C/T-13910 genotypes is a practical way of diagnosing adult type hypolactasia in an outpatient setting. Undiagnosed coeliac disease, should be actively screened and diagnosed in order to apply a gluten free diet and avoid the GI-symptoms and nutritional deficiencies. Cow’s milk hypersensitivity in the adult population is difficult to diagnose since the mechanism in which it is mediated is still unclear. Measuring of cow’s milk protein specific antibodies IgE, IgA or IgG do not correlate with subjective milk-related GI-symptoms.