Genetic variation in natural populations of the cereal cyst nematode (Heterodera avenae Woll.) submitted to resistant and susceptible cultivars of cereals

The purpose of the present work was to study the genetic characteristics of cereal cyst nematode (Heterodera avenae) populations re-established after the long-term use of resistant oat cultivars in field conditions. Population features were analyzed through fitness components and variation in enzymatic polymorphism (esterase and malate dehydrogenase loci). The longest (6 year) use of the same resistance genes (oat cv Panema) at high frequency (Rotation IB) led to the selection of a resistance-breaking pathotype and to a decrease in viability which suggested either a founder effect or a lower reproductive potential for the new pathotype. Analysis of esterase allelic frequencies led to the conclusions that: (1) the genetic constitution of this pathotype was different from the reference population maintained on the susceptible host (oat cv Peniarth), and (2) that the esterase locus may develop a disequilibrium linkage with loci involved in virulence. Random mating was recorded at the whole-field level but not always at the single-plant level, suggesting that cultivation practices such as annual ploughing could play a major role in homogenizing subpopulations developed in the vicinity of a plant. These phenomena showed that the long-term use of highly effective resistance could provok marked genetic modifications in populations. These risks should be taken into account when deciding strategies for optimal use of resistance genes in nematode management programs.

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The invasion and development of the cereal cyst-nematode, Heterodera avenae in the roots of autumn-and spring-sown cereals

Observations were made at 2 or 4 wk intervals from December to harvest on all stages of Heterodera avenae in winter oats growing on infested land. Second-stage larvae were present in all soil samples except on 5 and 20 July. Invasion and development of larvae was slow during winter. The nodal and seminal roots of winter oats were both heavily invaded by the nematode; larvae which invaded seminal roots tended to become male whereas those in nodal roots tended to become female. There was a small second invasion in August. Females were first observed on the roots of winter oats on 17 May, 214 days after the crop was sown and 62 days after the first fourth-stage larva was observed. The nodal roots of spring barley contained few H. avenae larvae whereas these roots were heavily invaded in winter wheat and oats. In spring barley the nodal roots were developing in June and July when few second-stage larvae were in the soil whereas in winter oats and wheat the nodal roots were growing rapidly in April when larvae were most numerous, and so were heavily invaded.     

Biology of the hop cyst nematode Heterodera humuli

In an intensively sampled English hop garden, cysts of Heterodera humuli were found in every sample taken. Most occurred in the top 15 cm of soil, decreasing in number by half with every 15 cm in depth. Numbers of second-stage juveniles fluctuated during the vegetative period in a pattern which indicated that more than one generation, possibly two to three, were produced. The generation time was about 1.5 months. In pot experiments under controlled conditions (20°C; 16 h light/day) the life cycle lasted 40 days. The optimal temperature was 20°C for the development of H. humuli in the roots of its host Humulus lupulus. Most H. humuli juveniles invaded hop roots at 15°C, but egg hatch was greatest at 20°C. In moist soil, second-stage juveniles survived for at least 54 days and thereafter invaded roots and reproduced.     

大豆胞囊线虫的休眠

以前研究发现,辽宁地区大豆生长期间及收获期土壤中胞囊孵出的二龄幼虫量很少,推测线虫卵的休眠与大豆生长时期或季节相关。为明确该地区大豆胞囊线虫的休眠特点,2002-2003年采用田间随机多点取样、室内分离及模拟自然条件孵化等方法对大豆胞囊线虫的休眠进行深入研究。结果表明:在生长季节,感病品种辽豆10根围土壤中的白色雌虫、卵囊及褐色的胞囊均可孵出二龄幼虫,且孵化持续时间较长,第21d仍有幼虫孵出,白色雌虫及卵囊内的卵孵化率高于褐色胞囊;不同作物对其根围土壤中胞囊内卵的孵化影响不大,寄主作物大豆、非寄主作物玉米根围及休闲地土壤中的胞囊在条件适宜均可孵出二龄幼虫;季节对胞囊内卵的孵化有较大的影响,出苗期孵化率最高,收获期最低,2周时平均1个胞囊孵出幼虫分别为83.8和9.7条;胞囊皮对线虫卵的孵化有显著的影响。表明沈阳地区大豆胞囊线虫在正常和逆境条件下均有部分卵表现休眠。     

Cloning and characterization of two neuropeptide genes from cereal cyst nematode, Heterodera avanae from India

The cereal cyst nematode, Heterodera avenae (Wollenweber, 1924) is one of the most important plant parasitic nematodes of cereals. It is an obligate sedentary endo parasite causing considerable crop losses in wheat, barley and oats worldwide. FMRFamide-like peptides (FLPs) play critical role as neurotransmitters or neuromodulators in the nervous system and proposed as one of the important targets for the plant parasitic nematode management. Therefore, for the first time we have cloned and characterized two neuropeptide genes (flp-12 and flp-16) from the cDNA library of feeding female of H. avenae. Sequence analysis of FLPs revealed that both the neuropeptides are closely related with the parasitic as well as free-living nematodes. The flp-12 contains putative 22 residue long signal peptide at N-terminal suggesting its association with extra-cellular functions, while flp-16 does not contain signal peptide. Besides this, we have found highly conserved motif KFEFIRF in flp-12 and RFGK motif in flp-16. These two flp genes could be interesting and potential targets for functional validation to explore their utility for designing management strategies.     

The mitochondrial genome of the soybean cyst nematode, Heterodera glycines

We sequenced the entire coding region of the mitochondrial genome of Heterodera glycines. The sequence obtained comprised 14.9 kb, with PCR evidence indicating that the entire genome comprised a single, circular molecule of approximately 21-22 kb. The genome is the most T-rich nematode mitochondrial genome reported to date, with T representing over half of all nucleotides on the coding strand. The genome also contains the highest number of poly(T) tracts so far reported (to our knowledge), with 60 poly(T) tracts ≥ 12 Ts. All genes are transcribed from the same mitochondrial strand. The organization of the mitochondrial genome of H. glycines shows a number of similarities compared with Radopholus similis, but fewer similarities when compared with Meloidogyne javanica. Very few gene boundaries are shared with Globodera pallida or Globodera rostochiensis. Partial mitochondrial genome sequences were also obtained for Heterodera cardiolata (5.3 kb) and Punctodera chalcoensis (6.8 kb), and these had identical organizations compared with H. glycines. We found PCR evidence of a minicircular mitochondrial genome in P. chalcoensis, but at low levels and lacking a noncoding region. Such circularised genome fragments may be present at low levels in a range of nematodes, with multipartite mitochondrial genomes representing a shift to a condition in which these subgenomic circles predominate.     

The global importance of the cereal cyst nematode (Heterodera spp.) on wheat and international approaches to its control

The Cereal Cyst Nematodes (CCNs) are a group of several closely related species which have been documented to cause economic yield loss on rainfed wheat production systems in several part of the world including North Africa, West Asia, China, India, Australia, America and several countries in Europe. The most commonly reported species is Heterodera avenae, however there are at least two other species H. filipjevi and H. latipons are implicated. It is well appreciated that plants under water and nutrient stress suffer greater yield loss. Control of CCNs requires maintaining nematode populations below economic thresholds. Chemicals are not environmentally sustainable or economic and the major emphasis on control has been with host genetic resistance applied with other integrated pest managent options. Unfortunately due to the number of species and pathotype variation genetic control of Cereal Cyst Nematode with plant resistance is complex. Turkey is one of the top ten wheat producers in the world and has identified these nematode as a major biotic constraint in their rainfed wheat systems. In 2001 a new joint intiative was established between CIMMYT International, the Turkish Ministry of Agriculture and (Ukurova University in Adana to understand i) the distribution of cereal nematodes on wheat; ii) assess the economic importance and improve our understanding of the population dynamics iii) culture, screen and assess known sources of resistance and identify new sources to both groups of nematodes; iv) integrate new sources of resistance into bread wheat cultivars for Turkey and International germplasm using conventional and molecular tools; v) investigate other integrated control options such as rotation and different wheat management strategies and finally vi) capacity build scientists to work in this important area. Some highlights of this work will be presented and the newly formed ICCNI - International Cereal Cyst Nematode Initative introduced.     

Root responses to cereal cyst nematode (Heterodera avenae) in hosts with different resistance genes

The development of cereal cyst nematode (CCN; Heterodera avenae ) induced syncytia in the host roots of infected resistant bread wheat ( Triticum aestivum cv. AUS10894), diploid wheat ( Aegilops tauschii ), barley ( Hordeum vulgare cv. Chebec and cv. Galleon) and in the susceptible wheat cv. Meering and barley cv. Clipper were studied over a period of 13 d. The resistance to CCN in these cereal plants is conferred by the resistance genes Cre1 in the wheat cv. AUS10894, Cre3 in A. tauschii , Ha2 in barley cv. Chebec and Ha4 in barley cv. Galleon. Anatomical observations were made on the development of the syncytia in CCN-infected wheat and barley roots, which carry each of these four sources of resistance genes. Accelerated development of the syncytia in resistant plants, especially in the barley cultivars, was observed. The sites of syncytia development in susceptible wheat and barley were also closely associated with the vascular tissues in the stele, but less so in the resistant plants. The syncytia in the infected susceptible wheat and barley were also metabolically active at day 13. By contrast, the syncytia of resistant wheat plants carrying the Cre1 or Cre3 genes remained extensively vacuolated and less metabolically active. In barley plants with the Ha2 or Ha4 genes, the syncytia appeared non-functional and in early stages of degeneration by day 13 after inoculation.     

Effect of different potting systems; inoculation time; nematode density and sources of cereal cyst nematode (Heterodera filipjevi) on juvenile penetration into wheat root system

Investigations were designed to optimize testing systems for screening wheat breeding lines for resistance to Heterodera filipjevi. The effects of: 1) plant potting systems 2) inoculum level and time of inoculation 3) and type of inoculum of H. filipjevi on detection accuracy were examined in growth chamber experiments in Turkey. The rate of nematode penetration in the highly susceptible variety Bezostaya was used as the base measurement of efficacy. The results showed that the highest level of penetration coupled with high level of germination was obtained in plastic tubes (13 cm long x 3 cm in diam.) when compared to both small flower pots (400 cm3) and smaller plastic tubes (10.2 cm long x 0.8 cm in diam.). The highest rate of nematode penetration into wheat root system was obtained by inoculating the seedlings with 1000 J2 per plant. However, inoculation with 200 J2 at sowing or 200 J2 at sowing plus an additional 200 J2 after germination improved percent penetration when compared to inoculation with 600 or 1000 J2/plant at sowing. The test on the optimum form of inoculum showed that inoculating the seedling with J2's gave the highest rate of nematode penetration over inoculum with eggs or cysts. The results of these experiments demonstrated that screening wheat for resistance can be optimized by raising the seedlings in plastic tubes and inoculating them with 400 J2.     

Effect of urea and certain NPK fertilizers on the cereal cyst nematode (Heterodera avenae) on wheat

Two outdoor pot experiments were conducted in two consecutive years under outdoor conditions during the wheat growing season in Saudi Arabia to determine the effects of urea and certain compound fertilizers (NPK), compared to the effects of the nematicide fenamiphos on the cereal cyst nematode (CCN), Heterodera avenae, and wheat growth. The results showed that all of the treatments, except the fertilizer diammonium phosphate (DAP), reduced the number of nematode cysts/root system and increased (P ⩽ 0.05) the dry weight of nematode-infected wheat plants. Fenamiphos and urea resulted in the best control, followed by the NPK fertilizers. The combined application of urea and fenamiphos resulted in the most significant effect in decreasing (P ⩽ 0.05) the number of cysts/root system and increasing (P ⩽ 0.05) the growth of nematode-infected wheat plants.     

Analysis of ascorbate peroxidase genes expressed in resistant and susceptible wheat lines infected by the cereal cyst nematode, Heterodera avenae

Changes in ascorbate peroxidase (APX) enzyme activity in response to nematode (Heterodera avenae) attack were studied in roots of three hexaploid wheat lines carrying Cre2, Cre5, or Cre7 nematode resistance genes and the susceptible Triticum aestivum cv. Anza. A spectrophotometric analysis was carried out with root extracts of infected plants 4, 7, 11, and 14 days after nematode inoculation using uninfected plant as control. APX induction in infected resistant genotypes was similar and higher than in the susceptible control. The introgression wheat/Aegilops ventricosa H-93-8 line, carrying the Cre2 gene, and its parental line H-10-15 as susceptible control were used to analyze whether this increase of activity was correlated with the induction of APX gene expression. Genes encoding cytosolic forms of APX were induced in roots of both lines in response to nematode infection. This induction took place both earlier and with greater intensity in the resistant line than in the susceptible one, and it was also higher in the root area at the site of nematode attachment.     

The effects of nematode resistant and susceptible spring oat cultivars and aldicarb on the cereal cyst nematode Heterodera avenae and yields in contrasting soil types

The effects of the cereal cyst-nematode, Heterodera auenae Woll. on resistant and susceptible oat cultivars, with and without aldicarb treatment, were compared on a clay-with-flints soil at Rothamsted and a loamy sand at Woburn. At both sites, when H. auenae was extremely scarce, yields were not further enhanced by aldicarb. At Rothamsted aldicarb increased yields by 48–72% when H. auenae averaged 10 eggs/g soil. At Woburn, aldicarb increased yields of both susceptible and resistant varieties by 80–90% with 20 eggs/g. The resistant varieties conferred yield benefits in the following oat crop equal to the residual effects of aldicarb applied before the previous crop, demonstrating that H. auenae was wholly responsible for the yield losses. Nematode resistant oats suffered as much or more damage from root invasion by H. auenae juveniles as the susceptible varieties but the resulting decrease in nematode numbers led to considerable yield improvements in the following year. At Woburn in 1977, when formalin was an added treatment, fewer females were infected by parasitic fungi and post-crop egg numbers were greater.     

A cereal cyst nematode (Heterodera avenae Woll.) resistance gene transferred from Aegilops triuncialis to hexaploid wheat

 The cereal cyst nematode (Heterodera avenae) is an important root parasite of common wheat. A high level of resistance was transferred to wheat from Aegilops triuncialis (TR lines) using the cross [(T. turgidum×Ae. triuncialis)×T. aestivum]. Low fertility (3–5 viable kernels per plant) was observed during the process but the surviving hybrid plants were highly vigorous. To obtain stable resistant lines further crosses to T. aestivum were performed. The resistance in TR lines seems to be transferred from the C genome of Ae. triuncialis (genomes CCUU). Ae. triuncialis was highly resistant to the two Spanish populations of H. avenae tested, as well as to four French races and two Swedish populations. The histological analysis showed a hypersensitive reaction in the roots of a resistant TR line inoculated with the Ha71 pathotype of H. avenae, whereas well-formed syncytia were observed in the roots of the susceptible control. Resistance to the H. avenae Ha71 pathotype seemed to be inherited as determined by a single dominant factor in the crosses between resistant TR lines and susceptible cultivars. Received: 11 November 1997 / Accepted: 9 December 1997     

Monoclonal antibodies to the soya bean cyst nematode, Heterodera glycines

In order to identify proteinaceous secretions involved in feeding of the soya bean cyst nematode, Heterodera glycines, over 1800 monoclonal antibodies (MAbs) were raised in four experiments using extracts of second stage juveniles and adult females as the immunogens. A rapid, indirect immunofluorescent screening procedure is described that enabled the antigens for 100 MAbs/day to be localised in juveniles. A similar immunofluorescent screening procedure for adult females was less successful due to the masking of the fluorophore by autofluorescence; additional approaches, including enzyme linked immunosorbent assays (ELISA), were necessary to aid in the screening process. The immunosuppressive drug, cyclophosphamide, had an effect on the range of antibodies obtained in two experiments. One was designed to favour antibodies specific to the hatched rather than the unhatched juvenile, whereas the second aimed to select those for the anterior rather than the posterior part of the adult female. A large number of MAbs showed well defined specificities and numbers are given in parentheses for those that recognised the following potential sites of secretion into the plant: the subventral pharyngeal glands (84), the dorsal pharyngeal gland (5), the amphidial pouches (10) and the excretory system (15). These MAbs will aid further work to define the role of nematode secretions in host invasion and the feeding process of this cyst nematode.     

Two-dimensional proteome reference maps for the soybean cyst nematode Heterodera glycines

2-DE reference maps of Heterodera glycines were constructed. After in-gel digestion with trypsin, 803 spots representing 426 proteins were subsequently identified by LC-MS/MS. Proteins with annotated function were further categorized by Gene Ontology. The results showed that proteins involved in metabolic, developmental and biological regulation processes were the most abundant.     

Studies of the cereal cyst-nematode, Heterodera avenae under continuous cereals, 1974–1978. I. Plant growth and nematode multiplication

Population changes of Heterodera avenae and crop growth in a sandy loam soil were studied from 1974 until 1978; the nematode decreased plant growth but failed in two of the years to multiply on susceptible hosts. Spring oats were the most heavily invaded cereal and produced the smallest shoots. Second-stage juveniles invaded cereal roots in decreasing numbers: spring oats > autumn oats > spring barley > spring wheat > autumn barley > autumn wheat. Numbers of females developing on the different cultivars were in a similar order. Most females developed on roots in 1976 despite poor crop growth in the severe drought. Numbers of H. avenae in soil treated with oxamyl (Vydate) at 8.8 kg/ha a. i. were less in all years except 1975. In the dry winter and spring of 1975/76 nematode multiplication was prevented in soil treated with oxamyl before drilling in the autumn. In all years large numbers of females were produced on the roots of all cultivars but in 1975 and 1978 nematode populations declined because few females survived to form cysts containing eggs and their fecundity was reduced. Numbers of cysts after harvest were not affected by formalin (38% formaldehyde) applied as a drench at 3000 litres/ha in 1977 but fecundity doubled in treated soil, and nematode multiplication increased from 3.8 × in untreated plots to 18.6 ×. When the plots were irrigated in 1978 numbers of cysts and fecundity increased in formalin treated soil resulting in an increase in multiplication from 0.3 × to 14.6 ×. Fungal parasites attacking H. avenae females and eggs are considered responsible for the poor multiplication of the nematode.     

Reproduction of the beet cyst nematode Heterodera schachtii Schm. on transformed root cultures of Beta vulgaris L.

Transformed hairy root cultures of Beta vulgaris L., grown in petri-dishes, were inoculated with a suspension of surface-sterilized larvae of the beet cyst nematode Heterodera schachtii Schm. Larvae developed into both male and female adults. Juvenile larvae were hatched from the newly-formed cysts, indicating that fertilization had occurred. Results of a glasshouse test showed that H. schachtii did not lose its pathogenicity after being cultured on transformed roots. This technique can be developed further for the mass-propagation of sedentary nematodes and for the in vitro storage of isclates.