Non-specific transient mutualism between the plant parasitic nematode, Bursaphelenchus xylophilus , and the opportunistic bacterium Serratia quinivorans BXF1, a plant-growth promoting pine endophyte with antagonistic effects

The aim of this study is to understand the biological role of Serratia quinivorans BXF1, a bacterium commonly found associated with Bursaphelenchus xylophilus, the plant parasitic nematode responsible for pine wilt disease. Therefore, we studied strain BXF1 effect in pine wilt disease. We found that strain BXF1 promoted in vitro nematode reproduction. Moreover, the presence of bacteria led to the absence of nematode chitinase gene (Bxcht-1) expression, suggesting an effect for bacterial chitinase in nematode reproduction. Nevertheless, strain BXF1 was unable to colonize the nematode interior, bind to its cuticle with high affinity or protect the nematode from xenobiotic stress. Interestingly, strain BXF1 was able to promote tomato and pine plant-growth, as well as to colonize its interior, thus, acting like a plant-growth promoting endophyte. Consequently, strain BXF1 failed to induce wilting symptoms when inoculated in pine shoot artificial incisions. This bacterium also presented strong antagonistic activities against fungi and bacteria isolated from Pinus pinaster. Our results suggest that B. xylophilus does not possess a strict symbiotic community capable of inducing pine wilt disease symptoms as previously hypothesized. We show that bacteria like BXF1, which possess plant-growth promoting and antagonistic effects, may be opportunistically associated with B. xylophilus, possibly acquired from the bacterial endophytic community of the host pine.

he genome and genetics of a high oxidative stress tolerant Serratia sp. LCN16 isolated from the plant parasitic nematode Bursaphelenchus xylophilus

Background: Pine wilt disease (PWD) is a worldwide threat to pine forests, and is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Bacteria are known to be associated with PWN and may have an important role in PWD. Serratia sp. LCN16 is a PWN-associated bacterium, highly resistant to oxidative stress in vitro, and which beneficially contributes to the PWN survival under these conditions. Oxidative stress is generated as a part of the basal defense mechanism used by plants to combat pathogenic invasion. Here, we studied the biology of Serratia sp. LCN16 through genome analyses, and further investigated, using reverse genetics, the role of two genes directly involved in the neutralization of H2O2, namely the H2O2 transcriptional factor oxyR; and the H2O2-targeting enzyme, catalase katA. Results: Serratia sp. LCN16 is phylogenetically most closely related to the phytosphere group of Serratia, which includes S. proteamaculans, S. grimessi and S. liquefaciens. Likewise, Serratia sp. LCN16 shares many features with endophytes (plant-associated bacteria), such as genes coding for plant polymer degrading enzymes, iron uptake/ transport, siderophore and phytohormone synthesis, aromatic compound degradation and detoxification enzymes. OxyR and KatA are directly involved in the high tolerance to H2O2 of Serratia sp. LCN16. Under oxidative stress, Serratia sp. LCN16 expresses katA independently of OxyR in contrast with katG which is under positive regulation of OxyR. Serratia sp. LCN16 mutants for oxyR (oxyR::int(614)) and katA (katA::int(808)) were sensitive to H2O2 in relation with wild-type, and both failed to protect the PWN from H2O2-stress exposure. Moreover, both mutants showed different phenotypes in terms of biofilm production and swimming/swarming behaviors. Conclusions: This study provides new insights into the biology of PWN-associated bacteria Serratia sp. LCN16 and its extreme resistance to oxidative stress conditions, encouraging further research on the potential role of this bacterium in interaction with PWN in planta environment.

Characterization of glutathione S transferases from the plant-parasitic nematode, Bursaphelenchus xylophilus

We have previously identified two secreted glutathione S-transferases (GST) expressed in the pharyngeal gland cell of Bursaphelenchus xylophilus, which are upregulated post infection of the host. This study examines the functional role of GSTs in B. xylophilus biology. We analysed the expression profiles of all predicted GSTs in the genome and the results showed that they belong to kappa and cytosolic subfamilies and the majority are upregulated post infection of the host. A small percentage is potentially secreted and none is downregulated post infection of the host. One secreted protein was confirmed as a functional GST and is within a cluster that showed the highest expression fold change in infection. This enzyme has a protective activity that may involve host defences, namely in the presence of terpenoid compounds and peroxide products. These results suggest that GSTs secreted into the host participate in the detoxification of host-derived defence compounds and enable successful parasitism.

Identification and characterization of parasitism genes from the pinewood nematode Bursaphelenchus xylophilus reveals a multilayered detoxification strategy.

The migratory endoparasitic nematode Bursaphelenchus xylophilus, which is the causal agent of pine wilt disease, has phytophagous and mycetophagous phases during its life cycle. This highly unusual feature distinguishes it from other plantparasitic nematodes and requires profound changes in biology between modes. During the phytophagous stage, the nematode migrates within pine trees, feeding on the contents of parenchymal cells. Like other plant pathogens, B. xylophilus secretes effectors from pharyngeal gland cells into the host during infection.We provide the first description of changes in the morphology of these gland cells between juvenile and adult life stages. Using a comparative transcriptomics approach and an effector identification pipeline, we identify numerous novel parasitism genes which may be important for the mediation of interactions of B. xylophilus with its host. In-depth characterization of all parasitism genes using in situ hybridization reveals two major categories of detoxification proteins, those specifically expressed in either the pharyngeal gland cells or the digestive system. These data suggest that B. xylophilus incorporates effectors in a multilayer detoxification strategy in order to protect itself from host defence responses during phytophagy.

Interview with Dr. Young-Ki Paik, President of the Human Proteome Organization (HUPO): Pharmacoproteomics and the approaching wave of “Proteomics Diagnostics”

Dr. Young-Ki Paik directs the Yonsei Proteome Research Center in Seoul, Korea and was elected as the President of the Human Proteome Organization (HUPO) in 2009. In the December 2009 issue of the Current Pharmacogenomics and Personalized Medicine (CPPM), Dr. Paik explains the new field of pharmacoproteomics and the approaching wave of “proteomics diagnostics” in relation to personalized medicine, HUPO’s role in advancing proteomics technology applications, the HUPO Proteomics Standards Initiative, and the future impact of proteomics on medicine, science, and society. Additionally, he comments that (1) there is a need for launching a Gene-Centric Human Proteome Project (GCHPP) through which all representative proteins encoded by the genes can be identified and quantified in a specific cell and tissue and, (2) that the innovation frameworks within the diagnostics industry hitherto borrowed from the genetics age may require reevaluation in the case of proteomics, in order to facilitate the uptake of pharmacoproteomics innovations. He stresses the importance of biological/clinical plausibility driving the evolution of biotechnologies such as proteomics,instead of an isolated singular focus on the technology per se. Dr. Paik earned his Ph.D. in biochemistry from the University of Missouri-Columbia and carried out postdoctoral work at the Gladstone Foundation Laboratories of Cardiovascular Disease, University of California at San Francisco. In 2005, his research team at Yonsei University first identified and characterized the chemical structure of C. elegans dauer pheromone (daumone) which controls the aging process of this nematode. He is interviewed by a multidisciplinary team specializing in knowledge translation, technology regulation, health systems governance, and innovation analysis.

Generating transgenic banana (cv. Sukali Ndizi) resistant to Fusarium Wilt

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi.

Natural and engineered plasmin inhibitors : applications and design strategies

Plasmin is the primary enzyme responsible for dissolution of fibrin in the circulatory system. Plasminogen, the zymogen of plasmin is expressed ubiquitously in the human body [1], with the predominant source being the liver [2, 3]. Plasminogen is produced as an 810 amino acid protein with a 19 amino acid leader peptide, which is cleaved during secretion to produce the mature 791 amino acid one-chain zymogen. This is converted to plasmin by cleavage of the Arg561 - Val562 scissile bond [4], resulting in an active protease consisting of two disulfide linked chains. The amino-terminal heavy chain (residues Glu1-Arg561) is comprised of a plasminogen/apple/nematode (PAN) domain [5] and five kringle domains of approximately equal size [6] while the light chain (residues Val562-Asn791) contains a serine protease domain homologous to trypsin with a catalytic triad comprising His603, Asp646 and Ser741 [7]. Both plasmin and plasminogen occur in two forms, full length and a Lys77-Lys78 activated variant produced through self catalysis (Figure 1). The former exists in a tight conformation through binding of Lys50 and/or Lys62 to kringle domain 5 [8, 9] while Lys78-plasminogen assumes a more relaxed conformation rendering it more susceptible to plasmin conversion [10, 11].

C. elegans RNA-dependent RNA polymerases rrf-1 and ego-1 silence Drosophila transgenes by differing mechanisms

Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis. © 2012 Springer Basel.

Slaving and release in co-infection control

Background Animal and human infection with multiple parasite species is the norm rather than the exception, and empirical studies and animal models have provided evidence for a diverse range of interactions among parasites. We demonstrate how an optimal control strategy should be tailored to the pathogen community and tempered by species-level knowledge of drug sensitivity with use of a simple epidemiological model of gastro-intestinal nematodes. Methods We construct a fully mechanistic model of macroparasite co-infection and use it to explore a range of control scenarios involving chemotherapy as well as improvements to sanitation. Results Scenarios are presented whereby control not only releases a more resistant parasite from antagonistic interactions, but risks increasing co-infection rates, exacerbating the burden of disease. In contrast, synergisms between species result in their becoming epidemiologically slaved within hosts, presenting a novel opportunity for controlling drug resistant parasites by targeting co-circulating species. Conclusions Understanding the effects on control of multi-parasite species interactions, and vice versa, is of increasing urgency in the advent of integrated mass intervention programmes.

Sustaining productivity of a Vertosol at Warra, Queensland, with fertilisers, no-tillage or legumes. 7. Yield, nitrogen and disease-break benefits from lucerne in a two-year lucerne–wheat rotation

Continuous cultivation and cereal cropping of southern Queensland soils previously supporting native vegetation have resulted in reduced soil nitrogen supply, and consequently decreased cereal grain yields and low grain protein. To enhance yields and protein concentrations of wheat, management practices involving N fertiliser application, with no-tillage and stubble retention, grain legumes, and legume leys were evaluated from 1987 to 1998 on a fertility-depleted Vertosol at Warra, southern Queensland. The objective of this study was to examine the effect of lucerne in a 2-year lucerne–wheat rotation for its nitrogen and disease-break benefits to subsequent grain yield and protein content of wheat as compared with continuous wheat cropping. Dry matter production and nitrogen yields of lucerne were closely correlated with the total rainfall for October–September as well as March–September rainfall. Each 100 mm of total rainfall resulted in 0.97 t/ha of dry matter and 26 kg/ha of nitrogen yield. For the March–September rainfall, the corresponding values were 1.26 t/ha of dry matter and 36 kg/ha of nitrogen yield. The latter values were 10% lower than those produced by annual medics during a similar period. Compared with wheat–wheat cropping, significant increases in total soil nitrogen were observed only in 1990, 1992 and 1994 but increases in soil mineralisable nitrogen were observed in most years following lucerne. Similarly, pre-plant nitrate nitrogen in the soil profile following lucerne was higher by 74 kg/ha (9–167 kg N/ha) than that of wheat–wheat without N fertiliser in all years except 1996. Consequently, higher wheat grain protein (7 out of 9 seasons) and grain yield (4 out of 9 seasons) were produced compared with continuous wheat. There was significant depression in grain yield in 2 (1993 and 1995) out of 9 seasons attributed to soil moisture depletion and/or low growing season rainfall. Consequently, the overall responses in yield were lower than those of 50 kg/ha of fertiliser nitrogen applied to wheat–wheat crops, 2-year medic–wheat or chickpea–wheat rotation, although grain protein concentrations were higher following lucerne. The incidence and severity of the soilborne disease, common root rot of wheat caused by Bipolaris sorokiniana, was generally higher in lucerne–wheat than in continuous wheat with no nitrogen fertiliser applications, since its severity was significantly correlated with plant available water at sowing. No significant incidence of crown rot or root lesion nematode was observed. Thus, productivity, which was mainly due to nitrogen accretion in this experiment, can be maintained where short duration lucerne leys are grown in rotations with wheat.

Host-delivered RNAi: An effective strategy to silence genes in plant parasitic nematodes

Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.

Development of key soil health indicators for the Australian banana industry

To improve the sustainability and environmental accountability of the banana industry there is a need to develop a set of soil health indicators that integrate physical, chemical and biological soil properties. These indicators would allow banana growers, extension and research workers to improve soil health management practices. To determine changes in soil properties due to the cultivation of bananas, a paired site survey was conducted comparing soil properties under conventional banana systems to less intensively managed vegetation systems, such as pastures and forest. Measurements were made on physical, chemical and biological soil properties at seven locations in tropical and sub-tropical banana producing areas. Soil nematode community composition was used as a bioindicator of the biological properties of the soil. Soils under conventional banana production tended to have a greater soil bulk density, with less soil organic carbon (C) (both total C and labile C), greater exchangeable cations, higher extractable P, greater numbers of plant-parasitic nematodes and less nematode diversity, relative to less intensively managed plant systems. The organic banana production systems at two locations had greater labile C, relative to conventional banana systems, but there was no significant change in nematode community composition. There were significant interactions between physical, chemical and nematode community measurements in the soil, particularly with soil C measurements, confirming the need for a holistic set of indicators to aid soil management. There was no single indicator of soil health for the Australian banana industry, but a set of soil health indicators, which would allow the measurement of soil improvements should include: bulk density, soil C, pH, EC, total N, extractable P, ECEC and soil nematode community structure.

Beyond chemical dependency for managing plant-parasitic nematodes: Examples from the banana, pineapple and vegetable industries of tropical and subtropical Australia

Plant-parasitic nematodes are important pests of horticultural crops grown in tropical and subtropical regions of Australia. Burrowing nematode (Radopholus similis) is a major impediment to banana production and root-knot nematodes (predominantly Meloidogyne javanica and M. incognita) cause problems on pineapple and a range of annual vegetables, including tomato, capsicum, zucchini, watermelon, rockmelon, potato and sweet potato. In the early 1990s, nematode control in these industries was largely achieved with chemicals, with methyl bromide widely used on some subtropical vegetable crops, ethylene dibromide applied routinely to pineapples and non-volatile nematicides such as fenamiphos applied up to four times a year in banana plantations. This paper discusses the research and extension work done over the last 15 years to introduce an integrated pest management approach to nematode control in tropical and subtropical horticulture. It then discusses various components of current integrated pest management programs, including crop rotation, nematode monitoring, clean planting material, organic amendments, farming systems to enhance biological suppression of nematodes and judicious use of nematicides. Finally, options for improving current management practices are considered.

Root-lesion nematodes (Pratylenchus thornei and P. neglectus): A review of recent progress in managing a significant pest of grain crops in northern Australia

Two species of root-lesion nematode (predominantly Pratylenchus thornei but also P. neglectus) are widespread pathogens of wheat and other crops in Australia's northern grain belt, a subtropical region with deep, fertile clay soils and a summer-dominant rainfall pattern. Losses in grain yield from P. thornei can be as high as 70% for intolerant wheat cultivars. This review focuses on research which has led to the development of effective integrated management programs for these nematodes. It highlights the importance of correct identification in managing Pratylenchus species, reviews the plant breeding work done in developing tolerant and resistant cultivars, outlines the methods used to screen for tolerance and resistance, and discusses how planned crop sequencing with tolerant and partially resistant wheat cultivars, together with crops such as sorghum, sunflower, millets and canaryseed, can be used to reduce nematode populations and limit crop damage. The declining levels of soil organic matter in cropped soils are also discussed with reference to their effect on soil health and biological suppression of root-lesion nematodes.