Some isolates of the nematophagous fungus Pochonia chlamydosporia promote root growth and reduce flowering time of tomato

The fungal parasite of nematode eggs Pochonia chlamydosporia is also a root endophyte known to promote growth of some plants. In this study, we analysed the effect of nine P. chlamydosporia isolates from worldwide origin on tomato growth. Experiments were performed at different scales (Petri dish, growth chamber and greenhouse conditions) and developmental stages (seedlings, plantlets and plants). Seven P. chlamydosporia isolates significantly (P < 0.05) increased the number of secondary roots and six of those increased total weight of tomato seedlings. Six P. chlamydosporia isolates also increased root weight of tomato plantlets. Root colonisation varied between different isolates of this fungus. Again P. chlamydosporia significantly increased root growth of tomato plants under greenhouse conditions and reduced flowering and fruiting times (up to 5 and 12 days, respectively) versus uninoculated tomato plants. P. chlamydosporia increased mature fruit weight in tomato plants. The basis of the mechanisms for growth, flowering and yield promotion in tomato by the fungus are unknown. However, we found that P. chlamydosporia can produce Indole-3-acetic acid and solubilise mineral phosphate. These results suggest that plant hormones or nutrient ability could play an important role. Our results put forward the agronomic importance of P. chlamydosporia as biocontrol agent of plant parasitic nematodes with tomato growth promoting capabilities.

Chitosan enhances parasitism of Meloidogyne javanica eggs by the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol.

Isolation and characterization of microorganisms with probiotic potential

Tese de mestrado em Microbiologia Aplicada, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016

Distribution of deep-sea meiobenthos in surface sediments off North Carolina

Observations on the ecology and distribution of meiofauna occurring on the outer continental shelf and continental slope at depths from 50 to 2500 m in the region where the Blake Plateau cuts across the North Carolina slope are reported. Total numbers of meiofauna ranged from 151/100 cm**3 of sediment at 400 m to 1196/100 cm**3 of sediment at 250 m. Sediments of the upper region (50-500 m) consisted of medium-sized calcareous sands with relatively low organic carbon contents, while the deeper sediments (600-2500 m) consisted of sandy silts and silts with organic carbon contents 6-10 times that of the shallower sediments. Two basic faunas appear to be present in the areas investigated; a shallow-water fauna extending from 50 to 500 m and a deep-water fauna from 800 to 2500 m. The shallow-water fauna consists of nematodes (the dominant taxon) and relatively large numbers of harpactacoid copepods, ostracods, benthic foraminifera, polychaetes, gastrotrichs and several other groups, while below 500 m only nematodes and foraminifera are present in large numbers, the latter being especially abundant between 800 and 2000 m. A major change in the meiofauna occurs on the Blake Plateau between the depths of approximately 400-500 m and 600-750 m where the composition of the sediment changes from sand to silty sand. From 50 m to 400-500 m gastrotrichs, turbellaria, tardigrades, kinorhynchs, halicarids, hydrozoans, gnathostomulids, lamellibranchs and cumaceans are commonly encountered; these groups are absent below 500 m. In addition, there are significant reductions in the numbers of harpactacoids, ostracods, nemerteans and polychaetes below 500 m. Examination of the nematode population also show faunal differences between the shallower sediments (50-500 m) and the deeper sediments (600-2500 m). High indices of affinity exist among the faunas between 50 and 500 m and among the faunas between 800 and 2500 m; the fauna at 600-750 m represents a transition between these two regions, but it is more closely related to the deep-water fauna. Changes in the distribution of both the total meiofuna and also the nematodes are highly correlated with changes in sediments composition and bottom water temperatures. It is suggested that changes in grain size and accompanying changes in sources of nutrition, which are the results of Gulf Stream and other current activity, are the dominant environmental factors influencing the meiofauna of the area.

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term nemepsins for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

A simple practical method for bulk and rapid extraction of free-living nematodes from marine and estuarine sediments

A simple, rapid method is described for the extraction of large numbers of free-living nematodes from estuarine sediments. This method does not physically or chemically alter or damage the nematodes, but instead relies on their downward movement through a filtering layer of double ply tissue paper and into aerated water-filled trays. Seven trials each with 10 trays kept at 25degreesC for an initial period of 24 h yielded 3985 live nematodes l(-1) (+/-511.5 standard deviation) of estuarine sediment, free of sediment and with minimal debris. Time effects were statistically significantly different, with the same 10 trays yielding another 1259 nematodes l(-1) (+/-413.4) when kept for a second period of 24 h at the same temperature. Temperature effects were also significant, and 7 trials each with 10 trays kept for 24 h at 20-21degreesC, produced a lower yield of 2160 nematodes l(-1) (+/-532.7) of sediment. The method is expected to be of use in nematode extractions from both estuarine and marine sediments.

Effect of Fusarium oxysporum f. sp. glycines and Sclerotium rolfsii on the pathogenicity of Meloidogyne incognita race 2 to soybean

Screenhouse studies were conducted to investigate the effects of Fusarium oxysporum f. sp. glycines and Sclerotium rolfsii on the pathogenicity of Meloidogyne incognita race 2 on soybean and the influence of the nematode on wilt incidence and growth of soybean. The interaction of each fungus with the nematode resulted in reduced shoot and root growth. Final nematode population was also reduced with concomitant inoculation of nematode and fungus or inoculation of fungus before nematode. While M. incognita suppressed wilt incidence in two nematode-susceptible cultivars of soybean (TGX 1485-2D and TGX 1440-IE), it had limited effect on wilt incidence in the nematode resistant cultivar of soybean (TGX 1448-2E). When F. oxysporum was inoculated with the nematode, the mean number of nematodes that penetrated soybean roots decreased by 75% in TGX 1448-2E, 68% in TGX 1485-1D and 65% in TGX 1440-1E. Similarly when the soil was treated with S. rolfsii, the number decreased by 78% in TGX 1448-2E, 77% in TGX 1485-1D and 68% in TGX 1440-1E. The nematode did not develop beyond second-stage juvenile in TGX-1448-2E.

CemaT1 is an active transposon within the Caenorhabditis elegans genome

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and T-C transposons, with a distribution thus far limited to a few invertebrate species. In the nematode Caenorhabditis elegans, there are eight copies of CemaT1 that are predicted to encode a functional transposase, with five copies being >99% identical. We present evidence, based on searches of publicly available databases and on PCR-based mobility assays, that the CemaT1 transposase is expressed in C. elegans and that the CemaT transposons are capable of excising in both somatic and germline tissues. We also show that the frequency of CemaT1 excisions within the genome of the N2 strain of C. elegans is comparable to that of the Tc1 transposon. However, unlike T-C transposons in mutator strains of C elegans, maT transposons do not exhibit increased frequencies of mobility, suggesting that maT is not regulated by the same factors that control T-C activity in these strains. Finally, we show that CemaT1 transposons are capable of precise transpositions as well as orientation inversions at some loci, and thereby become members of an increasing number of identified active transposons within the C. elegans genome. (C) 2004 Elsevier B.V. All rights reserved.

Prevalence, intensity and risk factors for soil-transmitted helminth infection in a South Indian fishing village

A study of the prevalence, intensity and risk factors for soil-transmitted helminth infection was undertaken among school children aged 5-9 years attending a primary school in the fishing village in Peda Jalaripet, Visakhapatnam, South India. One hundred and eighty nine (92.6%) of 204 children were infected with one or more soil transmitted helminth parasites. The predominant parasite was Ascaris lumbricoides (prevalence of 91%), followed by Trichuris trichiura (72%) and hookworm (54%). Study of age-specific prevalence and intensity of infection revealed that the prevalence and intensity of A. lumbricoides infection was higher among younger children than older children. While aggregation of parasite infection was observed, hookworm infection was more highly aggregated than either A. lumbricoides or T. trichiura. Multivariate analysis identified parental occupation, child's age and mother's education as the potential risk factors contributing to the high intensity of A. lumbricoides infection. Children from fishing families with low levels of education of the mother had the highest intensity of A. lumbricoides infection. As the outcome of chemotherapy programs to control soil transmitted helminth infection is dependant on the dynamics of their transmission, there is a need for further studies to better define the role of specific factors that determine their prevalence, intensity and aggregation in different epidemiological settings. (C) 2004 Elsevier B.V. All rights reserved.

The Caenorhabditis briggsae genome contains active CbmaT1 and Tcb1 transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.

In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni

Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Bioactivity of Fusarium oxysporum f. sp glycines and Sclerotium rolfsii filtrates on egg hatching, survival and infectivity of juveniles of Meloidogyne incognita race 2

The effects of culture filtrates of Fusarium oxysporum and Sclerotium rolfsii on egg hatching and juvenile survival of Meloidogyne incognita in vitro and impact of these filtrates on infectivity of M. incognita were investigated on soybean seedlings. Five- and 10-day-old filtrates of F. oxysporum caused 65 and 54% egg-hatching inhibition, while that of S. rolfsii caused 61 and 49% inhibition, respectively. Juveniles of M. incognita died within 6 days when incubated in 5-day-old filtrate of F. oxysporum, while the similar filtrate of S. rolfsii caused 100% juvenile mortality on the fifth day. Filtrates reduced root galling, egg population, number of adult females in soybean plants at harvest and also soil population. Culture filtrates could be used as source of biological nematicides.

Angiostrongrylus cantonensis as a cause of cerebrospinal disease in a yellow-tailed black cockatoo (Calyptorhynchus funereus) and two tawny frogmouths (Podargus strigoides)

A captive yellow-tailed black cockatoo (Calyptorhynchus funereus) and 2 free-living tawny frogmouths (Podargus strigoides), both native Australian species, were presented with neurologic signs including depression and pelvic limb paresis and paralysis. Despite supportive treatment, all 3 birds died or were euthanatized. On histologic examination, sections of metastrongyloid nematode larvae were found in the central nervous system of all 3 birds, whereas intact larvae, identified as Angiostrongylus cantonensis, were recovered from the brain and spinal cord of 2 birds. Angiostrongylus cantonensis, the rat lungworm. has an obligatory migratory phase through the host's central nervous system, which can cause severe pathologic lesions. Natural infections in accidental hosts have been documented only in mammals, and to our knowledge, angiostrongyliasis in avian hosts has not been previously reported.

Analysing diversity in sugarcane resistance gene analogues

As resistance genes have been shown to contain conserved motifs and cluster in many plant genomes, the identification of resistance gene analogues can be used as a strategy for both the discovery of DNA markers linked to disease resistance loci and the map-based cloning of disease resistance genes. Sugarcane suffers from many important diseases and an analysis of resistance gene analogues offers a means to identify DNA markers linked to resistance loci. However, sugarcane has the most complex genome of any crop plant and initially it is important to understand the extent of resistance gene analogue diversity in the sugarcane genome before genetic analysis. We review herein how more than 100 expressed sequence tags with homology to different resistance genes have been identified in sugarcane with many mapped as single-dose restriction fragment length polymorphism markers. Importantly, some of these resistance gene analogues have been shown to be linked to disease resistance genes or disease quantitative trait loci. In an attempt to more efficiently analyse additional resistance gene analogues in sugarcane, we report on experiments aimed at investigating the molecular diversity of several resistance gene analogue families using a modified form of a technique termed Ecotilling. Using Ecotilling, we were able to rapidly detect single nucleotide polymorphisms in fragments amplified by PCR from four different resistance gene analogue families, SoRP1D, SoPTO, SoXa21 and SoHs1pro-1. An analysis of a diverse set of sugarcane varieties, including modern sugarcane cultivars and several S. officinarum and S. spontaneum clones, indicated that all amplicons, apart from SoHs1pro-1, contained significant polymorphism within the gene region studied. However, a comparison among these sugarcane clones, including between the parents of two sugarcane mapping populations, indicated that most polymorphisms were multi-dose, not single-dose, preventing their genetic map location or association with disease susceptibility or resistance from being determined.