Ecology and Control of Cereal Cyst Nematode (Heterodera avenae) in Southern Australia

The ecology and control of cereal cyst nematode in southern Australia is reviewed. The wide distribution of Heterodera avenae in Victoria and South Australia is due largely to movement of cysts by wind during dust storms. The fungus Rhizoctonia solani frequently is associated with the nematode in a disease complex in wheat, and disease symptoms are most severe on lighter or well structured soils. Crop rotations which include periods of fallow, or of nonhost crop reduce population levels of H. avenae and improve yields. Early-sown crops (April-May) are less severely damaged than late-sown crops (June-July). The resowing of damaged wheat crops or the application of nitrogenous fertilizers rarely improve grain yields. ''Katyil,'' the world''s first wheat cultivar bred specifically with resistance to H. avenae, has been released in Victoria. Chemical control of the nematode in cereals is now commercially feasible, and five nematicides are registered for use by growers.     

Fungal Parasites of the Cereal Cyst Nematode Heterodera avenae in Southern Sweden

Fungal parasites of the cereal cyst nematode Heterodera avenae were isolated from four sites in southern Sweden. In all, 15 different fungi were isolated from different stages of the nematode life cycle. Among the egg parasites, Verticillium chlamydosporium was common in young cysts on roots, whereas an unidentified species of Verticillium (Verticillium sp. 1) was the dominating species in cysts from soil, especially if the soll had been stored for 8-12 months. V. chlamydosporium was frequently isolated from eggs in cysts from soil, when analyzed shortly after sampling. Verticillium sp. 1 is distinct from V. chlamydosporium because it does not produce dichtyo-chlamydospores in the aerial mycelium and because it grows at 6 C where V. chlamydosporium fails to grow. Paecilomyces lilacinus, Microdochium bolleyi, Cylindrocarpon sp., and several nonsporulating fungi were also isolated from eggs in cysts from soil. Between 10 and 20% of the eggs in cysts collected in the field were infected with fungi. In a pot test between < 1 and 29%, with a mean of 13%, of females on roots became infected, always by Nematophthora gynophila. Resting spores of N. gynophila extracted directly from field soil, collected at the four sites, varied from 3 to 49 spores/gram of air dried soil.     

Cereal Cyst Nematode (Heterodera avenae) on Oats. II. Early Root Development and Nematode Tolerance

The effect of Heterodera avenae infestation on early seminal and lateral root growth was examined in four oat genotypes differing in tolerance to H. avenae. Recently emerged seminal roots were inoculated with a range of H. avenae larval densities, then transferred a hydroponic system to remove the effect of later nematode penetration on root development. Intolerance to H. avenae was assessed in terms of impairment of seminal root extension resulting in fewer primary lateral roots emerging from the seminal root below the zone of juvenile penetration. Tolerant plants infested with H. avenae had longer lateral root systems than infested intolerant plants. The decline in lateral root growth below the penetration zone was partly offset by increased growth above. This did not contribute to tolerance, however, as there were no differences between cultivars for this feature. Nematodes induced earlier nodal root emergence in all cultivars. Nodal root development was most advanced on the most tolerant cultivar.     

Molecular Cloning and Characterization of Two Genes Encoding Tryptophan Decarboxylase from Aegilops variabilis with Resistance to the Cereal Cyst Nematode (Heterodera avenae) and Root-Knot Nematode (Meloidogyne naasi)

Tryptophan decarboxylase (TDC), which catalyzes the conversion of Trp to tryptamine, provides a common backbone for many secondary metabolites, and is important in defending plants from abiotic stress such as pathogen infection and insect attack. In this study, we cloned two TDC genes, AeVTDC1 and AeVTDC2, from Ae. variabilis accession No. 1 with resistance to cereal cyst nematode (CCN) and root-knot nematode (RKN). AeVTDC1 and AeVTDC2 encode polypeptides of 510 and 518 amino acids, respectively, and both have a pyridoxal phosphate attachment site and specific catalytic residues. Comparative analyses of gene structure and amino acid motifs revealed that TDCs are highly conserved crossing the analyzed species in monocots and dicots. Phylogenetic analysis indicated that AeVTDCs were closer to TDCs of wheat, Ae. tauschii, Triticum urartu, Brachypodium distachyon, and Hordeum vulgare. Their functions and temporal and spatial expression patterns were investigated. Moreover, AeVTDC1 and AeVTDC2 exhibited different expression responses to the phytohormones abscisic acid, salicylic acid, and methyl jasmonate, suggesting that they may function differently in response to biotic and abiotic stresses. The inhibition of TDC activity with S-αFMT resulted in susceptibility of Ae. variabilis to CCN and RKN, suggesting that TDCs may play important roles in resistance to nematodes.

     

World Dissemination of the Cereal-Cyst Nematode (Heterodera avenae) and Its Potential as a Pathogen of Wheat

World distribution of the cereal-cyst nematode is herein reviewed. It is suggested that Heterodera avenae originated in Europe and has been widely disseminated, largely by the activities of Man but also by wind movement of cysts. So far, it may not have spread to some major wheat-growing regions of the New World, but a non-friable soil structure limits population level and disease. Yield loss could result from the introduction of new cultivars to developing countries where H . avenae has not been detected or where existing cultivars possess tolerance.     

New Species of Cyst Nematode Heterodera pakistanensis (Nematoda: Heteroderidae) Attacking Wheat in Pakistan

Heterodera pakistanensis n. sp., described and illustrated from roots of common wheat (Triticum aestivum) from Sukkur, Sind, Pakistan, belongs to the goettingiana group. It is most closely related to H. cyperi Golden, Rau &Cobb, 1962, H. raskii Basnet & Jayaprakash, 1984, and H. mothi Khan &Husain, 1965. Second-stage juveniles (J2) can be distinguished from H. cyperi J2 by an areolated lateral field with four incisures and shorter stylet, whereas cysts are separated by a more elongated vulva slit and the conspicuous structure of the underbridge. It differs from H. raskii by having four areolated lateral lines in J2, smaller female lemon-shaped cyst, shorter fenestra length and width, conspicuous underbridge, and distinct anus with a high cuticular pattern 40-45 μm from posterior end. It also differs from H. mothi by the presence of four areolated lateral lines in J2 and absence of vulva denticles and bullae.     

Partial Resistance to Heterodera avenae in Wheat Lines with the 6M v Chromosome from Aegilops ventricosa

Lines of wheat with the 6M^v chromosome from Aegilops ventricosa display partial resistance to both pathotypes Hal2 and Ha41 of Heterodera avenae. With either pathotype, the effect of this alien chromosome on cyst production, size, and fecundity was expressed in resistance tests. Partial resistance of five 6M^v(6D) substitution lines varied according to the intrinsic cyst-forming capacity of the nematode pathotypes and the recipient germplasms. Such partial resistance can be utilized in wheat breeding lines for integrated management of the cereal cyst nematode.     

Genetic Analysis of Esterase Polymorphism in the Soybean Cyst Nematode, Heterodera glycines

The genetic basis of esterase polymorphism in Heterodera glycines was investigated through controlled matings and analysis of F₁ and F₂ progeny. Three nematode lines, each fixed for a different esterase phenotype, were isolated and purified through repeated directional selection and inbreeding. Each phenotype was characterized by its distinct pair of closely spaced bands of esterase activity. Single-female single-male crosses were conducted according to a modified agar-plate mating technique. F₁ progeny were homogeneous, exhibiting both parental esterase phenotypes (codominant heterozygotes) but no hybrid bands. Approximately 1,500 F₂ progeny segregated in a 1:2:1 ratio for expression of the esterase phenotypes of the female parental line, the heterozygote, and the male parental line. Apparently the three esterase phenotypes correspond to three codominant alleles of a single esterase locus. Reciprocal crosses gave similar results, suggesting no maternal inheritance.     

Identification of Candidate Genes Related to Stem Development in Brassica napus Using RNA-Seq

Plant Molecular Biology Reporter - Plant stems are involved in supporting the entire plant body, thus having an important effect on the yield of oilseed rape. The current understanding of the...     

Bacteria Associated with Cysts of the Soybean Cyst Nematode (Heterodera glycines)

The soybean cyst nematode (SCN), Heterodera glycines, causes economically significant damage to soybeans (Glycine max) in many parts of the world. The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops. To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means. Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst. Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 ± 0.5) × 10⁵ bacteria (mean ± standard deviation) with various cellular morphologies. Filamentous fungi were also observed. Live-dead staining indicated that the majority of cyst bacteria were viable. The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature. Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition. Bands shared by all eight samples included the actinobacterium genera Actinomadura and Streptomyces. A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium. These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence. Thirty phylotypes were detected, with the collection dominated by Lysobacter and Variovorax spp. This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents.     

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 ({"type":"entrez-nucleotide","attrs":{"text":"KU877266","term_id":"1008457416","term_text":"KU877266"}}KU877266), Ha-far-2 ({"type":"entrez-nucleotide","attrs":{"text":"KU877267","term_id":"1008457418","term_text":"KU877267"}}KU877267), Hf-far-1 ({"type":"entrez-nucleotide","attrs":{"text":"KU877268","term_id":"1008457420","term_text":"KU877268"}}KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.     

Scanning Electron Microscopy of Vulval Cones of Heterodera glycines and Three Related Cyst Nematode Species

Vulval cones of four closely related Heterodera species - H. glycines (races 1-5), H. lespedezae, H. schachtii, and H. trifolii - were examined using scanning electron microscopy. Numbers of dorsal and ventral radial ridges, total radial ridges, perineal ridges, and preanal ridges were useful in differentiating the five races of H. glycines and the other three species. Most of the populations differed significantly (P < 0.01) using the Waller-Duncan k-ratio t-test for mean separation of the five characters. H. glycines races 2, 4, and 5 were most similar. H. schachtii and H. trifolii were most dissimilar to each other and to H. lespedezae and H. glycines. Two additional qualitative characters were also useful in differentiating the populations. The shallower, shorter radial ridges of H. glycines provided a basis for separation from the other three species. Width and smoothness of the perineal ridges were useful in differentiating both races and species.     

Molecular Characterization of a Soybean Cyst Nematode (Heterodera glycines) Homolog of unc-87

In Caenorhabditis elegans the unc-87 gene encodes a protein that binds to actin at the I band and is important in nematodes for maintenance of the body-wall muscle. Caenorhabditis elegans mutant phenotypes of unc-87 exhibit severe paralysis in larvae and limp paralysis in the adult. We cloned and characterized a full-length cDNA representing a Heterodera glycines homolog of the unc-87 gene from C. elegans that encodes a protein that contains a region of seven repeats similar to CLIK-23 from C-elegans and has 81% amino acid identity with that of C. elegans unc-87 variant A. In the EST database clones labeled "unc-87'''' encode mainly the 3'' portion of unc-87, while clones labeled "calponin homolog OV9M'''' contain mainly DNA sequence representing the 5'' and middle transcribed regions of unc-87. A 1770 nucleotide cDNA encoding H. glycines unc-87 was cloned and encodes a predicted UNC-87 protein product of 375 amino acids. The expression of unc-87 was determined using RT-PCR and, in comparison to its expression in eggs, unc-87 was expressed 6-fold higher in J2 juveniles and 20-fold and 13-fold (P = 0.05) higher in nematodes 15 and 30 days after inoculation, respectively. In situ hybridization patterns confirmed the expression patterns observed with RT-PCR.     

Development of PrimeTime-Real-Time PCR for Species Identification of Soybean Cyst Nematode (Heterodera glycines Ichinohe, 1952) in North Carolina

Soybean cyst nematode (SCN) is an obligate, sedentary parasite that is a major pathogen of soybean and accounts for an estimated 1 billion dollars in production losses annually in the United States of America. This paper describes the development of a real-time PCR method for rapid, sensitive, species-specific and accurate identification of SCN alone or on mixed populations with other nematodes in North Carolina. The 83-bp DNA fragment of PrimeTime-real-time PCR was designed based on a 477-bp-SCN-SCAR marker previously proved to be SCN-specific. A total of 44 populations including cyst forming nematodes (Heterodera glycines, H. fici, H. schachtii, H. trifolii, Cactodera weissi, Globodera tabacum, Meloidodera floridensis and other unidentified cyst nematodes) and non-cyst forming nematodes (Ditylenchus dipsaci, Meloidogyne incognita and Xiphinema chambersi) were tested in this study, all SCN populations are tested positive and non-SCN populations negative. This assay for the detection and identification has been successfully applied for testing a single SCN cyst, a 2^nd-stage-SCN juvenile, a single SCN egg, up to ten SCN cysts, a 10-fold dilution of a single 2^nd-stage-SCN juvenile and 20-fold dilution of one SCN cyst. The assay is not SCN-race specific. It gave an accurate positive result when SCN is mixed with other cyst species. Also, nematode universal primers/probes for real-time PCR amplification as a nematode endogenous control to detect the presence of 18S ribosomal RNA (rRNA) gene were employed in this assay, so that a SCN-negative sample can be tested to exclude false negative. This method will be very useful for a broad range of research programs as well as the regulatory response and management of SCN in North Carolina and other region of the southeastern U.S.A.     

Heterodera raskii n. sp. (Heteroderidae: Tylenchina), a Cyst Nematode on Grass, from Hyderabad,India

Heterodera raskii n. sp. is described and illustrated from specimens collected from roots of bulb grass, Cyperus bulbosus, in Hyderabad, India. The new species belongs to the ''goettingiana'' group and differs from closely related H. cyperi by the elongate ovoid shaped cysts and females, greater fenestral length, width, vulval slit, and absence of egg sac. The stylet knob shape was round in second-stage juveniles and posteriorly sloping in females and males of H. raskii n. sp., while it was anteriorly directed in second-stage juveniles and spherical in females and males of H. cyperi.