Elegans Eggs Research Articles

Interclutch variability in egg characteristics in two species of rail: Is maternal identity encoded in eggshell patterns?

Maternal signatures are present in the eggs of some birds, but quantifying interclutch variability within populations remains challenging. Maternal assignment of eggs with distinctive appearances could be used to non-invasively identify renesting females, including hens returning among years, as well as to identify cases of conspecific brood parasitism. We explored whether King Rail (Rallus elegans) eggs with shared maternity could be matched based on eggshell pattern. We used NaturePatternMatch (NPM) software to match egg images taken in the field in conjunction with spatial and temporal data on nests. Since we had only a small number of marked breeders, we analyzed similar clutch images from a study of Eurasian Common Moorhens (Gallinula chloropus chloropus) with color-banded breeders for which parentage at many nests had been verified genetically to validate the method. We ran 66 King Rail clutches (n = 338 eggs) and 58 Common Moorhen clutches (n = 364 eggs) through NPM. We performed non-metric multidimensional scaling and permutational analysis of variance using the best egg match output from NPM. We also explored whether eggs could be grouped by clutch using a combination of egg dimensions and pattern data derived from NPM using linear discriminant analyses. We then scrutinized specific matches returned by NPM for King Rail eggs to determine whether multiple matches between the same clutches might reveal maternity among nests and inform our understanding of female laying behavior. To do this, we ran separate NPM analyses for clutches photographed over several years from two spatially distant parts of the site. With these narrower datasets, we were able to identify four instances where hens likely returned to breed among years, four likely cases of conspecific brood parasitism, and a within-season re-nesting attempt. Thus, the matching output was helpful in identifying congruent egg patterns among clutches when used in conjunction with spatial and temporal data, revealing previously unrecognized site fidelity, within-season movements, and reproductive interference by breeding females. Egg pattern data in combination with nest mapping can be used to inform our understanding of female reproductive effort, success, and longevity in King Rails. These methods may also be applied to other secretive birds and species of conservation concern.

Designing a Novel CRISPR/Cas9 Endogenous Tag of the Amph-1 Gene for Treatment Research Purposes

Amphiphysin 1, or amph-1, is a c. elegans ortholog of the genes amphiphysin (AMPH) and bridging integrator 1 (BIN 1) present in humans. Amph-1 is located primarily in the endomembrane system of the cell. Its protein count is highly affected by diseases under dementia and carcinoma. To gain further understanding of amph-1 protein activity in response to such diseases, this research centers around the creation of a CRISPR/Cas9 design plan to inject modified plasmids into c. elegans eggs and produce specimens available for endogenous activity research. An N20 sequence and 5’ and 3’ homology arms were located near the N-terminus, where a bioluminescent protein tag would be inserted. A plasmid dedicated to synthesizing the Cas9 protein and a second plasmid carrying the genetic code for bioluminescent protein are then modified using amph-1 genetic code as a base template. Following silent mutation edits to the N20 sequence, this plasmid design has potential to be developed in labs for depper research into the amph-1 gene behavior.

A Facile and Modified Scheme for Synchronization and Isolation of Nematode Eggs

Nematodes are common pests that damage agricultural crop plants. Some of them are beneficial while others are parasitic and harmful to plants, animals and humans. Several in vitro studies have aimed to develop chemicals to kill parasitic nematodes, while others have been conducted to use beneficial nematodes as biocontrol agents. However, the preparation of large quantities of nematode eggs in a laboratory setting is challenging. Traditional egg isolation protocols involve the use of sieves to filter eggs or the use of chemicals that can be harmful to nematodes while isolating the eggs. Our method utilizes 1.5 × 1.5 cm sized chunks of bacterial or fungal feed to lure nematodes. A subsequent gentle washing of the consumed chunk with distilled water provides a rapid and straightforward method of collecting eggs in 6-well polystyrene plates and removing unwanted nematodes. Approximately 4000 Bursaphelenchus xylophilus eggs from a fungal chunk and 2400 Caenorhabditis elegans eggs from a bacterial chunk were obtained when tested. This study shows a protocol for the isolation of eggs and synchronization of nematode stages that is relatively straightforward, rapid, eco-friendly, and efficient. The protocol also provides a chemical-free and a reliable, simple means of separating eggs from adults and induces the synchronization of nematodes based on the simple concept that gravid nematodes can be provoked to lay eggs by providing additional feed.

Post-Translational Modifications in Oocyte Maturation and Embryo Development.

Mammalian oocyte maturation and embryo development are unique biological processes regulated by various modifications. Since de novo mRNA transcription is absent during oocyte meiosis, protein-level regulation, especially post-translational modification (PTM), is crucial. It is known that PTM plays key roles in diverse cellular events such as DNA damage response, chromosome condensation, and cytoskeletal organization during oocyte maturation and embryo development. However, most previous reviews on PTM in oocytes and embryos have only focused on studies of Xenopus laevis or Caenorhabditis elegans eggs. In this review, we will discuss the latest discoveries regarding PTM in mammalian oocytes maturation and embryo development, focusing on phosphorylation, ubiquitination, SUMOylation and Poly(ADP-ribosyl)ation (PARylation). Phosphorylation functions in chromosome condensation and spindle alignment by regulating histone H3, mitogen-activated protein kinases, and some other pathways during mammalian oocyte maturation. Ubiquitination is a three-step enzymatic cascade that facilitates the degradation of proteins, and numerous E3 ubiquitin ligases are involved in modifying substrates and thus regulating oocyte maturation, oocyte-sperm binding, and early embryo development. Through the reversible addition and removal of SUMO (small ubiquitin-related modifier) on lysine residues, SUMOylation affects the cell cycle and DNA damage response in oocytes. As an emerging PTM, PARlation has been shown to not only participate in DNA damage repair, but also mediate asymmetric division of oocyte meiosis. Each of these PTMs and external environments is versatile and contributes to distinct phases during oocyte maturation and embryo development.

A Simple Nematode Infection Model for Studying Candida albicans Pathogenesis.

Candida albicans is an opportunistic fungal pathogen and a model organism to study fungal pathogenesis. It exists as a harmless commensal organism and member of the healthy human microbiome, but can cause life-threatening mucosal and systemic infections. A model host to study C.albicans infection and pathogenesis is the nematode Caenorhabditis elegans. C.elegans is frequently used as a model host to study microbial-host interactions because it can be infected by many human pathogens and there are also close morphological resemblances between the intestinal cells of C.elegans and mammals, where C.albicans infections can occur. This article outlines a detailed methodology for exploiting C.elegans as a host to study C.albicans infection, including a C.elegans egg preparation protocol and an agar-based C.elegans killing protocol to monitor fungal virulence. These protocols can additionally be used to study C.albicans genetic mutants in order to further our understanding of the genes involved in pathogenesis and virulence in C.albicans and the mechanisms of host-microbe interactions. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of Caenorhabditis elegans eggs Support Protocol 1: Freezing and recovering Caenorhabditis elegans Support Protocol 2: Making superfood agar and OP50 plates Basic Protocol 2: Caenorhabditis elegans/Candida albicans agar killing assay Support Protocol 3: Constructing a worm pick.

Hydric environmental effects on turtle development and sex ratio

Experimental and field studies of different turtle species suggest that moisture influences embryonic development and sex ratios, wetter substrates tend to produce more males, and drier substrates produce more females. In this study, we used Trachemys scripta elegans to test the effect of moisture on embryonic development and sex ratios. T. s. elegans eggs were incubated under different temperature and moisture regimes. We monitored embryonic development until stage 22 (after sex determination) and, for the first time, we estimated sex ratios using a male-specific transcriptional molecular marker, Sox9. Among treatments, we found differences in developmental rates, egg mass, and sex ratio. Embryos developed slowly in cooler and wetter sand substrate while water uptake by the eggs was significantly greater on wetter substrates. Developmental differences were due to moisture interacting with temperature where increased water content of the sand resulted in temperatures that were 2–3°C lower than air temperatures. The coolest and the wettest substrates produced 100% males compared to 42% males from the warmest and driest treatment. Further, we found that embryonic growth appears to be more sensitive to temperature at earlier stages of development and to moisture at later stages. This study shows how moisture may change the incubation conditions inside nests by changing the temperature experienced by eggs, which affects development, growth and sex ratios. The results of this study highlight the importance of including moisture conditions when predicting embryo growth and sex ratios and in developing proxies of embryonic development.

Measuring the Effect of Chemicals on the Growth and Reproduction of <em>Caenorhabditis elegans</em>

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Exposure of C. elegans eggs to a glyphosate-containing herbicide leads to abnormal neuronal morphology

Recent data demonstrate that chronic exposure of Caenorhabditis elegans (C. elegans) to a high-use glyphosate-containing herbicide, Touchdown (TD), potentially damages the adult nervous system. It is unknown, however, whether unhatched worms exposed to TD during the egg stage show abnormal neurodevelopment post-hatching. Therefore, we investigated whether early treatment with TD leads to aberrant neuronal or neurite development in C. elegans. Studies were completed in three different worm strains with green fluorescent protein (GFP)-tagged neurons to facilitate visual neuronal assessment. Initially, eggs from C. elegans with all neurons tagged with GFP were chronically exposed to TD. Visual inspection suggested decreased neurite projections associated with ventral nerve cord neurons. Data analysis showed a statistically significant decrease in overall green pixel numbers at the fourth larval (L4) stage (*p<0.05). We further investigated whether specific neuronal populations were preferentially vulnerable to TD by treating eggs from worms that had all dopaminergic (DAergic) or γ-aminobutyric acid (GABAergic) neurons tagged with GFP. As before, green pixel number associated with these discrete neuronal populations was analyzed at multiple larval stages. Data analysis indicated statistically significant decreases in pixel number associated with DAergic, but not GABAergic, neurons (***p<0.001) at all larval stages. Finally, statistically significant decreases (at the first larval stage, L1) or increases (at the fourth larval stage, L4) in superoxide levels, a developmental signaling molecule, were detected (*p<0.05). These data suggest that early exposure to TD may impair neuronal development, perhaps through superoxide perturbation. Since toxic insults during development may late render individuals more vulnerable to neurodegenerative diseases in adulthood, these studies provide some of the first evidence in this model organism that early exposure to TD may adversely affect the developing nervous system.

Developmental abnormality induced by strong static magnetic field in Caenorhabditis elegans.

Understanding the effects of strong static magnetic fields (SMFs) on living organisms is significant in health risk assessment, but underlying mechanisms are largely unknown. In the present study, we determined developmental abnormalities induced by 8.5Tesla (T) SMFs in a well-established in vivo model organism, Caenorhabditis elegans (C. elegans). Exposure of C. elegans eggs to 8.5 T SMF resulted in a time-dependent lifespan decrease, whereas only slight changes were observed upon exposure to 5 T SMF. Although SMF exposure did not alter brood size, development rate and stages were significantly modified by 8.5 T SMF. Germ cell apoptosis dramatically increased upon exposure to 8.5 T SMF in adult worms, as confirmed by ced-3 and ced-4 mutants, and could be prevented by concurrent treatment with a free radical scavenger, dimethyl sulfoxide. Compared to wild-type worms, shorter lifespan and greater numbers of apoptotic cells were observed in abnormal methyl viologen sensitivity-1 (mev-1(kn1)) nematodes with increased sensitivity to oxidative damage. Furthermore, exposure to 8.5 T SMF increased expression of superoxide dismutase-3 (sod-3), which is thought to protect against oxidative stress. However, 8.5 T SMF had minimal effects on lifespans of daf-2 and daf-16 mutants, which have compromised insulin/IGF-1 (insulin-like growth factors-1) mediated signaling pathways; this finding was consistent with the expression of these genes in wild-type worms. Our results indicate that developmental toxicity induced by strong SMF in C. elegans is mediated by oxidative stress and may be regulated by the insulin-like receptor pathway.

Experimental evidence of size/age-biased infection of Biomphalaria glabrata (Pulmonata: Planorbidae) by an incompatible parasite species: Consequences for biological control

Because the digenetic trematode Plagiorchis elegans can elicit a rapid, severe and permanent suppression of the reproductive output in the snail Biomphalaria glabrata, it is considered as a potential biological control agent of human schistosomiasis. This assumption however is derived from laboratory experiments that are poor approximations of what occurs in a natural ecosystem. In order to recreate conditions that resemble those found in nature, we exposed B. glabrata as individual populations composed of a young, juvenile and adult snails to various concentrations of P. elegans eggs to assess the probability of encountering the parasite eggs by the different snail sizes/age groups. We demonstrated that within populations composed of different size/age classes, larger/older snails displayed the negative effects typical of exposure to P. elegans, whereas smaller individuals appeared relatively unaffected, particularly at lower levels of exposure. These findings coupled with the difficulty of producing large quantities of parasite eggs suggest that P. elegans has limited efficiency as a biological control agent of human schistosomiasis.

Using Caenorhabditis elegans as a model organism for evaluating extracellular signal-regulated kinase docking domain inhibitors

We have recently identified several novel ATP-independent inhibitors that target the extracellular signal-regulated kinase-2 (ERK2) protein and inhibit substrate phosphorylation. To further characterize these compounds, we describe the use of C. elegans as a model organism. C. elegans is recognized as a versatile and cost effective model for use in drug development. These studies take advantage of the well characterized process of vulva development and egg laying, which requires MPK-1, the homolog to human ERK2. It is shown that treatment of C. elegans eggs or larvae prior to vulva formation with a previously identified lead compound (76) caused up to 50% reduction in the number of eggs produced from the adult worm. In contrast, compound 76 had no effect on egg laying in young adult or adult worms with fully formed vulva. The reduction in egg laying by the test compound was not due to effects on C. elegans life span, general toxicity, or non-specific stress. However, compound 76 did show selective inhibition of phosphorylation of LIN-1, a MPK-1 substrate essential for vulva precursor cell formation. Moreover, compound 76 inhibited cell fusion necessary for vulva maturation and reduced the multivulva phenotype in LET-60 (Ras) mutant worms that have constitutive activation of MPK-1. These findings support the use of C. elegans as a model organism to evaluate the selectivity and specificity of novel ERK targeted compounds.

Effects of Developmental Exposure to Ethanol on Caenorhabditis elegans

We investigated the effects of chronic ethanol exposure on physical development, reproduction, and life expectancy of Caenorhabditis elegans, a microscopic nematode worm. It has a small nervous system of 302 neurons and a short lifespan of 2 to 3 weeks. In this study, the worms were chronically exposed to varying concentrations of ethanol for different periods of their life: for their entire lifespan, during larval development only, and during adulthood only. In addition, the worms were exposed to ethanol acutely during different stages of embryonic development. Chronic exposure to ethanol during larval development temporarily delayed physical growth, slowed development, delayed the onset of reproductive maturity, and decreased both reproductive fecundity and longevity. Chronic exposure to ethanol beginning when worms completed development and reached reproductive maturity resulted in reduced C. elegans body length, decreased reproductive fecundity, and life expectancy. Finally, acute embryonic exposure of C. elegans eggs to high concentrations of ethanol at different stages of development resulted in a lower probability of exposed eggs hatching into larval worms depending on when eggs were exposed during development. Furthermore, some of the worms that did hatch displayed distinct physical dysmorphologies as a consequence of acute ethanol exposure during embryonic development. These data suggest that exposing C. elegans to ethanol during critical development periods results in characteristic phenotypic outcomes. Thus, C. elegans offers a novel model for exploring the mechanisms by which ethanol exposure affects development.

Molecular cloning and characterization of the Caenorhabditis elegans α1,3-fucosyltransferase family

The genome of Caenorhabditis elegans encodes five genes with homology to known alpha1,3 fucosyltransferases (alpha1,3FTs), but their expression and functions are poorly understood. Here we report the molecular cloning and characterization of these C. elegans alpha1,3FTs (CEFT-1 through -5). The open-reading frame for each enzyme predicts a type II transmembrane protein and multiple potential N-glycosylation sites. We prepared recombinant epitope-tagged forms of each CEFT and found that they had unusual acceptor specificity, cation requirements, and temperature sensitivity. CEFT-1 acted on the N-glycan pentasaccharide core acceptor to generate Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-Asn. In contrast, CEFT-2 did not act on the pentasaccharide acceptor, but instead utilized a LacdiNAc acceptor to generate GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc, which is a novel activity. CEFT-3 utilized a LacNAc acceptor to generate Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc without requiring cations. CEFT-4 was similar to CEFT-3, but its activity was enhanced by some divalent cations. Recombinant CEFT-5 was well expressed, but did not act on available acceptors. Each CEFT was optimally active at room temperature and rapidly lost activity at 37 degrees C. Promoter analysis showed that CEFT-1 is expressed in C. elegans eggs and adults, but its expression was restricted to a few neuronal cells at the head and tail. We prepared deletion mutants for each enzyme for phenotypic analysis. While loss of CEFT-1 correlated with loss of pentasaccharide core activity and core alpha1,3-fucosylated glycans in worms, loss of other enzymes did not correlate with any phenotypic changes. These results suggest that each of the alpha1,3FTs in C. elegans has unique specificity and expression patterns.

Evaluation of a combination of sodium hypochlorite and polyhexamethylene biguanide as an egg wash for red-eared slider turtles (Trachemys scripta elegans) to suppress or eliminate Salmonella organisms on egg surfaces and in hatchlings

To evaluate a combination of 2 nonantibiotic microbicide compounds, sodium hypochlorite (NaOCl) and polyhexamethylene biguanide (PHMB), as a treatment to suppress or eliminate Salmonella spp from red-eared slider (RES) turtle (Trachemys scripta elegans) eggs and hatchlings. 2,738 eggs from 8 turtle farms in Louisiana. Eggs were randomly sorted into 3 or, when sufficient eggs were available, 4 treatment groups as follows: control, pressure-differential egg treatment with NaOCl and gentamicin, NaOCl and PHMB bath treatment, and pressure-differential egg treatment with NaOCl and PHMB. Bacterial cultures were performed from specimens of eggs and hatchlings and evaluated for Salmonella spp. RES turtle eggs treated with NaOCl and PHMB as a bath (odds ratio [OR], 0.2 [95% confidence interval (CI), 0.1 to 0.3]) or as a pressure-differential dip (OR, 0.01 [95% CI, 0.001 to 0.07]) or with gentamicin as a pressure-differential dip (OR, 0.1 [95% CI, 0.06 to 0.2]) were significantly less likely to have Salmonella-positive culture results than control-group eggs. Concern over reptile-associated salmonellosis in children in the United States is so great that federal regulations prohibit the sale of turtles that are < 10.2 cm in length. Currently, turtle farms treat eggs with gentamicin solution. Although this has reduced Salmonella shedding, it has also resulted in antimicrobial resistance. Results of our study indicate that a combination of NaOCl and PHMB may be used to suppress or eliminate Salmonella spp on RES turtle eggs and in hatchlings.

412 分配バックプロジェクション法を利用した細胞の3次元形態観察法の確立とその線虫卵への応用(OS4-3:細胞と機械工学:計測とモデリングの最前線,オーガナイズドセッション4:細胞と機械工学:計測とモデリングの最前線)

412 分配バックプロジェクション法を利用した細胞の3次元形態観察法の確立とその線虫卵への応用(OS4-3:細胞と機械工学:計測とモデリングの最前線,オーガナイズドセッション4:細胞と機械工学:計測とモデリングの最前線)

A method for obtaining quantities of Caenorhabditis elegans eggs

A method for obtaining quantities of Caenorhabditis elegans eggs

Effects of Geldanamycin on Hatching and Juvenile Motility in Caenorhabditis elegans and Heterodera glycines

Several Streptomyces species are known to produce metabolites that inhibit plant pathogens. One such compound is geldanamycin (GA), a benzoquinone ansamycin originally isolated from Streptomyces hygroscopicus. We examined the effect of geldanamycin on egg hatch and juvenile motility in Caenorhabditis elegans and in two populations of the plant-parasitic nematode Heterodera glycines. When C. elegans eggs were exposed to geldanamycin, both hatch and motility were reduced by GA doses between 2 and 50 microg/ml. The H. glycines inbred populations TN17 and TN18 exhibited low dose stimulation of hatch and motility, whereas levels occurring at higher GA doses were at or below control levels. These experiments represent the first demonstration of geldanamycin effects in C. elegans and H. glycines and suggest that the heat shock chaperone Hsp90, the known molecular target of geldanamycin, may be involved in nematode egg hatch and motility. This study also indicates that geldanamycin-producing strains of Streptomyces may be useful as biocontrol agents for nematodes.

Asymmetric Division: A Kinesin for Spindle Positioning

The meiotic spindles of animal eggs move to extremely asymmetric positions, close to the cell cortex. A recent paper has identified a motor complex that may move the meiotic spindle toward the cortex in Caenorhabditis elegans eggs.

The effect of incubation temperature on egg survival, hatchling traits and embryonic use of energy in the blue-tailed skink, Eumeces elegans

Abstract Eggs of the skink, Eumeces elegans were incubated at 24, 27, 30 and 33°C to assess the effect of temperature on hatchling traits and embryonic use of energy. The mean incubation periods of eggs incubated at 24, 27, 30 and 33°C were 44.1, 28.0, 23.6 and 20.0 days, respectively. The mortality of embryos incubated at these temperatures did not differ significantly. The mean snoutvent lengths of hatchlings from 30 and 33°C were smaller than those of hatchlings from 24°C. In contrast, the body masses of hatchlings were not affected by temperature. Incubation temperature can also modify limb lengths of hatchlings. The fore and hind limb lengths of hatchlings from 24°C were longer than those of hatchlings from 33°C. However, tail length and head size (length and width) for hatchlings from different temperatures were similar. The locomotor capacity of hatchlings incubated at 33°C was completely destroyed, whereas that of hatchlings from the other three temperatures did not differ significantly in spite of the existence of differences in hatchling size. Moreover, incubation temperature affected the allocation of energy between carcass and yolk sac in the hatchling, although the overall conversion of energy from eggs to hatchlings was not influenced by temperature. Hatchlings from 30 and 33°C had a less developed carcass and more energy in yolk sac than those from 24°C. Thus, we can conclude that 33°C is not suitable for the incubation of E. elegans eggs, due to damage to the embryo.

Cuticular disruption and mortality of Caenorhabditis elegans exposed to culture filtrate of Byssochlamys nivea Westling

AbstractA strain of a Byssochlamys nivea, isolated from saline mud in Western Australia as a part of statewide survey of soil fungi for nematophagous activity, was evaluated for its effect on nematodes. Culture filtrate of the fungus grown on potato dextrose broth for 7 days caused structural changes in the cuticle, aggregation of individuals, and mortality of Caenorhabditis elegans. In addition, the culture filtrate completely inhibited hatching of C. elegans eggs. Exudates from agar colonies also caused cuticular disruption and mortality of C. elegans. The cuticular disruption observed, not reported in nematodes before, was initiated in the labial region and spread towards the posterior region of the nematode within 10 min of application. This reaction occurred only in live nematodes. Cuticular disruption and mortality caused by the culture filtrate varied according to growth conditions. The active compound(s) in the culture filtrate were thermostable (100°C for 1 h); however freezing the culture filtrate (-20°C for 2 days) eliminated the activities, as did dialysis (&lt;14 000 molecular weight). Cuticular disruption and mortality were also observed when the nematode was exposed to culture filtrates of two other strains of B. nivea supplied by CBS, The Netherlands. The culture filtrate also inhibited in vitro growth of the plant-pathogenic fungi Fusarium oxysporum, Gaeumannomyces graminis var. tritici, Phytophthora cinnamomi, Pythium irregulare and Rhizoctonia solani.