Heterodera Carotae Research Articles

The carrot cyst nematode Heterodera carotae: a major plant-parasitic nematode requiring more investigation

Summary The present study reviews the knowledge to date on the carrot cyst nematode Heterodera carotae, which has become the primary threat to the carrot sector following the withdrawal of the last chemical soil fumigants in Europe. A keyword co-occurrence network was used to structure this review into five research areas: i) biology and epidemiology of H. carotae; ii) molecular identification and phylogeny; iii) population genetics and genomics; iv) control methods and strategies; and v) root exudates and hatching stimulation. Our findings indicate that H. carotae is an under-studied plant-parasitic nematode species, with several identified alternative control methods that require further investigation.

A diagnostic gap to fill: Development of molecular tools to distinguish the cyst-forming nematode species Heterodera carotae and Heterodera cruciferae

The carrot cyst nematode, Heterodera carotae, is an important pest causing qualitative and quantitative production losses across several countries worldwide. In culture crops, Heterodera carotae and Heterodera cruciferae, can be found in mixture and are virtually unrecognizable from a morphological point of view. However, only H. carotae is able to develop on carrot. To monitor carrot production and set up suitable H. carotae control plans, simple and reliable diagnostic methods to detect and identify these two species are needed. In this study, we developed and successfully tested two real-time PCR protocols (SYBR Green singleplex and TaqMan multiplex real-time PCRs) against both species: H. carotae and H. cruciferae. Using two specific sets of primers, we managed, whatever the methods, to distinguish the two species. Moreover, we showed a higher specificity using the TaqMan assays but a better sensitivity with the SYBR Green assays. In this study, we are proposing two useful and reliable diagnostic tools to enable farmers and scientists to rapidly detect two morphologically and genetically closely related species and supervise agricultural management.

Lure and starve: Host root exudates to suppress field populations of cyst nematodes

Plant parasitic nematodes are a burden to global food security, accounting for substantial yield losses in agricultural production worldwide. Increasing concerns over the environment and health issues have led to diminishing control options at the disposal of agricultural producers. Lure and starve, a strategy that is based on stimulating hatching of second stage juveniles (J2) of some cyst forming plant parasitic nematodes in the absence of their host plant seems a promising approach to maintain sustainability in crop production. Here we examined in-situ hatching of encysted eggs of the potato cyst nematode Globodera pallida and the carrot cyst nematode Heterodera carotae following exogenous applications of host root exudates in repeated field experiments during autumn and spring seasons. Population densities (viable J2 g−1 of soil) were assessed prior and post-application of root exudates. Results showed hatching induction of up to 83 % for G. pallida and 54 % for H. carotae in field plots drenched with their respective host root exudates. Spring season experiments were characterised by limited soil moisture and did not reduce nematode population densities for G. pallida. The potential for host root exudates in suppressing field population of cyst nematode in lure and starve strategies looks promising for both cyst nematode species investigated.

Occurrence and distribution of cyst nematodes, Heterodera spp. (Tylenchida: Heteroderidae) associated with black cabbage, Brassica oleracea var. acephala L. (Brassicales: Brassicaceae) in the Eastern Black Sea Region of Türkiye

This study was conducted during 2021-2022 to detect and determine distribution and population of cyst nematodes, Heterodera spp. (Tylenchida: Heteroderidae) in black cabbage Brassica oleracea var. acephala L. (Brassicales: Brassicaceae) production areas of the Eastern Black Sea Region of Türkiye. For it, a total of 77 samples were taken from 53 districts belonging to the Artvin, Giresun, Ordu, Rize, and Trabzon provinces in the region. Soil samples were taken from around the root of the kale plants. Nematodes were extracted by using the centrifugal flotation technique. The nematodes were identified using morphological features and molecular analysis based on Polymerase Chain Reaction (PCR) method. For molecular analysis, the ribosomal DNA region including the gene region of 28S ribosomal RNA (rRNA) (ITS1, 5.8S, ITS2) was amplified using primer sets TW81/AB28. Additionally, a species-specific primer set (Car-F/Car-R) covering the Cytochrome Oxidase I (cox1) region of mitochondrial DNA (mtDNA) was used. As a result of the analysis, cyst nematodes Heterodera cruciferae Franklin, 1945, Heterodera carotae Jones, 1950 and Heterodera fici Kirjanova, 1954 species were identified in the kale production areas in the region. Heterodera carotae is the first record of the cyst nematode species in Türkiye. Heterodera cruciferae, H. carotae, and H. fici were detected from the total collected soil samples at 16.9%, 3.9%, and 1.3% relative frequency, respectively. Among all, Giresun was the most infected province with 35.3% infection rate, followed by Trabzon with 26.3%, Ordu with 21.1% and Rize with 13.3%.

Genome Announcement: The Draft Genome of the Carrot Cyst Nematode Heterodera Carotae.

Heterodera carotae, the carrot cyst nematode, is a significant pest affecting carrot globally. Here we present the draft genome of H. carotae, which was generated from short read libraries from Illumina HiSeq technology, and the corresponding genome annotation.

Integrirani pristup zaštiti mrkve od dva značajna štetnika – mrkvine muhe i mrkvine cistolike nematode

Carrots are among the top ten vegetable crops in the world, with an annual world production of about 428 million tonnes grown on about 11.5 million hectares. The investment per unit area is not high and with good cultivation techniques yields of up to 50 t/ha can be achieved, together with major economic benefits from winter cultivation. During the growing season, carrots are susceptible to numerous pests. The most important are the carrot root fly (Psila rosae, Fabricius), whose root-feeding fly larvae reside in the soil or plant tissue, and the carrot cyst nematode (Heterodera carotae Jones), which is responsible for the decline of carrots in many countries and requires strict sanitation measures if crops are infected. As insecticides become less available in Europe, the appropriate use of non-chemical methods in integrated pest management (IPM) needs to be emphasised, highlighting the importance of developing new tolerant varieties. This review addresses the approaches to monitor and predict the occurrence of two important carrot pests and the main control measures according to the strategies of IPM.

Impact of the genetic diversity of three cyst nematodes on the effectiveness of root exudates to induce hatching

Cyst nematodes are sedentary endoparasites of plants which cause important economic losses worldwide. New nematode control measures are needed since the removal of effective chemical nematicides from the market due to their negative impact on environment and human health. Induced hatching of second-stage juveniles in the absence of host plant using root exudates, also named “suicide hatching”, could be a sustainable alternative method to reduce nematode population densities in infested soils. Unfortunately, biocontrol methods are often less effective in agricultural fields than in laboratory or greenhouse and this could be due to, among other reasons, a different susceptibility of nematode populations to root exudate stimulation. Testing this intra-specific variability would help to anticipate any drop in the efficiency of this new biocontrol strategy. A selection of root exudates from different plant species, maximizing the hatching level highlighted in a previous study, was tested on the hatching of representative populations of the genetic diversity for Globodera pallida, Heterodera schachtii and Heterodera carotae. Results showed significant differences between populations for given nematode species but not correlated with the genetic structure. Overall, root exudates tested provide a high level of hatching of these three nematode species. Surprisingly, the root exudate from broccoli induces hatching of European populations of the potato cyst nematode G. pallida.

Characterization of Heterodera carotae from the Tarlton area, South Africa, and determination of its life cycle under field conditions: a baseline study

Characterization of Heterodera carotae from the Tarlton area, South Africa, and determination of its life cycle under field conditions: a baseline study

Hatching Induction of Cyst Nematodes in Bare Soils Drenched With Root Exudates Under Controlled Conditions.

Cyst nematodes account for substantial annual yield losses in crop production worldwide. Concerns over environmental and health issues due to the use of chemical nematicides mean alternative sustainable and integrated solutions are urgently required. Hatch induction of encysted eggs in the absence of host plants, i.e., ‘suicide-hatching,’ could be a sustainable alternative in reducing population densities of cyst nematodes in infested soils. Here we examined in situ hatching of encysted eggs of Globodera pallida, Heterodera carotae, and Heterodera schachtii at varying soil depths, following exogenous applications of host root exudates in repeated glasshouse experiments. Cysts were retrieved 30 or 43 days post-incubation depending on the nematode species and assessed for hatching rates relative to the initial number of viable eggs per cyst. Hatching of the potato cyst nematode G. pallida depended on both soil moisture and effective exposure to root exudates, and to a lesser extent on exudate concentration. The carrot cyst nematode H. carotae had over 75% hatched induced by root exudate irrespective of the concentration, with better hatch induction at 20 cm as compared with 10 cm soil depth. Hatching of the beet cyst nematode H. schachtii largely depended on the soil moisture level at constant temperature, rather than the type or concentration of root exudates applied. As a conclusion, exogenously applied host root exudates may play a major role in inducing in situ hatch of encysted eggs of potato and carrot cyst nematodes in the absence of host plant under favorable soil temperature/moisture conditions. To improve such strategy, the characterization of chemical profiles of the root exudate composition and field validation are currently ongoing.

Evidence of strong gene flow among French populations of the carrot cyst nematode Heterodera carotae

The recent ban of the most efficient chemical nematicides has left growers without methods for controlling the carrot cyst nematode Heterodera carotae. This phytoparasitic nematode species has a very narrow host range and causes severe crop losses in the main carrot‐growing regions worldwide. The development of alternative means of management of H. carotae is thus essential, and knowledge is required about the adaptive abilities of H. carotae, which mainly depend on gene flow among populations. The goal of this study was to describe the genetic structure of H. carotae populations at the spatial scale of the main infested French carrot‐producing region, i.e. Lower Normandy, and to disentangle the causes of the heterozygote deficit in this polyvoltine species. Microsatellite genotyping of populations collected at both the plant and field scales showed that: (i) the heterozygote deficit is mainly due to substructure; and (ii) there is strong gene flow among populations, leading to low FST and to no clear genetic structure at the spatial scale explored here. Soil transport through both agricultural machinery and the transport of leek seedlings is probably responsible for the very strong H. carotae migration among fields and production areas. Measures should be considered to limit the passive spread of H. carotae.

Microsatellite markers reveal two genetic groups in European populations of the carrot cyst nematode Heterodera carotae

The cyst nematode Heterodera carotae, which parasitizes carrot roots, has been recorded in many countries in Europe (Italy, The Netherlands, Switzerland, France, Denmark, …), in South Africa and in North America (Canada, USA). To date, there is a lack of knowledge about the genetic structure of the populations of this economically important nematode. The aim of this work was to study the structuration of the genetic diversity of the carrot cyst nematode at the European scale. We have developed a set of thirteen polymorphic microsatellite markers and used it to genotype seventeen European populations of H. carotae coming from France, Switzerland, Italy, Denmark and one non-European population from Canada. As previously showed for other cyst nematode species, the H. carotae populations were characterised by a strong heterozygote deficit. A Bayesian clustering analysis revealed two distinct genetic clusters, with one group located in the north of Europe and a second one located in the south of Europe. Moreover, our results highlighted rather limited gene flow at small spatial scale and some events of long distance migration. This first investigation of the genetic diversity of H. carotae populations would be useful to develop sustainable control strategies.

First Report of Carrot Cyst Nematode Heterodera carotae in Mexico: Morphological, Molecular Characterization, and Host Range Study.

During 2008 to 2016 in several nematological surveys in the Tepeaca Valley, Puebla, Mexico, carrot cyst nematode, Heterodera carotae was found parasitizing carrots, Daucus carota . The nematode was present in 61% of the sampled fields with high population densities, causing severe carrot yield losses in the Tepeaca Valley. The aim of this work was to study morphology, morphometrics, host range, and molecular characterization of the nematode. The morphological and morphometric characterization was made using light and scanning electron microscopy of the second stage juveniles, females, males and cysts, and the host range study, was performed with nine different plants from five families. The molecular identification was made by sequencing and analysing the ITS rRNA and partial COI genes. It was shown that using presently available molecular tools it is not possible to make an accurate differentiation of H. carotae from H. cruciferae . The host range test allowed to distinguish these species from each other. Our study showed that male stylet length is longer for H. carotae compared with that for H. cruciferae . This is an example of importance of combination of several methods for the correct identification of cyst nematodes. To our knowledge, this is the first report of H. carotae in Mexico.

Integrative diagnosis of carrot cyst nematode (Heterodera carotae) using morphology and several molecular markers for an accurate identification

Cyst nematodes obtained from commercial carrot fields in Ontario (Canada) and northern and southern Italy were subjected to morphological and molecular examination. Morphology of cyst cone tops, males and second-stage juveniles (J2) indicated the nematode species was the Carrot Cyst Nematode (CaCN), Heterodera carotae. The sequence of the Internal Transcribed Spacer (ITS), D2-D3 region of the 28S gene of ribosomal RNA, cytochrome oxidase I of mitochondrial DNA (coxI), and a heat shock protein gene (hsp90), from single cysts were also examined. Sequences of ITS and D2-D3 placed all the nematodes with Heterodera carotae and other Heterodera spp. belonging to the Goettingiana group in the same clade. The novel nine coxI sequences obtained also clustered in a well-supported phylogenetic clade for H. carotae. Similarly, the six new hsp90 sequences of H. carotae generated in this study were placed in a well-supported clade (PP = 1.00) together with other two sequences of H. carotae from Greece. Restriction Fragment Length Polymorphism (RFLP) of ITS-PCR products gave a restriction pattern for RsaI different than H. carotae but the other 6 restriction patterns were similar as described in former research. A diagnostic conventional PCR method was developed based on a primer set to be specific for H. carotae using coxI sequence. These primers were also used in real time PCR to generate a melt curve specific to H. carotae. Limit of detection for CaCN in conventional PCR reaction was a single J2.

EFFICACY OF DIMETHYL DISULFIDE (DMDS) IN THE CONTROL OF THE ROOT-KNOT NEMATODE MELOIDOGYNE INCOGNITA AND THE CYST NEMATODE HETERODERA CAROTAE ON CARROT IN FIELD CONDITION IN ITALY

ISHS VIII International Symposium on Chemical and Non-Chemical Soil and Substrate Disinfestation EFFICACY OF DIMETHYL DISULFIDE (DMDS) IN THE CONTROL OF THE ROOT-KNOT NEMATODE MELOIDOGYNE INCOGNITA AND THE CYST NEMATODE HETERODERA CAROTAE ON CARROT IN FIELD CONDITION IN ITALY

Nematicidal potential of Brassicaceae

Brassicaceae Burnett (syn. Cruciferae A. L. de Jussieu) include many important economic plants used as edibile or ornamental. They are commonly known as the “mustard” plant family due to the sharp, potent flavour of their main metabolites, the glucosinolates (GLSs) which contain sulfur. Glucosinolates coexist in vivo with glycosylated thioglucosidases, myrosinase(s), responsible of their hydrolysis with the production of bioactive cognate isothiocyanates (ITC). GLSs and ITCs function as defence bioactive metabolites against plant pathogens, insects and herbivores. The present review paper focus on GLSs role as bionematicides. The current knowledge on the efficacy of these phytochemicals against the most common phytoparasitic nematodes affecting crops of agriculture importance such as tomato, potato and grapevine is reported. Data from our ongoing research on the in vitro biocidal activity of glucosinolate extracts, and their main components, against the virus-vector nematode Xiphinema index Thorne & Allen and the carrot cyst nematode Heterodera carotae Jones are also described.

Control of plant parasitic nematodes with active saponins and biomass from Medicago sativa

Medicago sativa L., alfalfa, is the most known plant species within the Medicago genus. The plant has been extensively studied for its content of saponins, mainly consisting of triterpene glycosides of medicagenic acid, possessing several biological properties including a biocidal activity on different soil microorganisms. Phytoparasitic nematodes are responsible for heavy economic damages to numerous agricultural crops and, due to their large distribution, they are among the most difficult crop pests to control. Attention on environmental safety and human and animal health has led to the progressive dismission of many synthetic formulations for the control of those pests and to the search of alternative strategies, including the use of natural metabolites from plants. Saponins from M. sativa may be good candidates for natural nematicide formulations, as in our in vitro studies the saponin mixtures from M. sativa tissues have been found effective in vitro against the virus-vector nematode Xiphinema index, the root-knot nematode Meloidogyne incognita and the potato cyst parasite, Globodera rostochiensis. A structure–activity relationship among saponins and related prosapogenins and sapogenin, respectively, has also been analyzed. The nematicidal efficacy differed among the three assayed nematode species, G. rostochiensis being the most susceptible to the active compounds from alfalfa. The in vitro results were also confirmed by experiments in potting mixes infested by M. incognita or G. rostochiensis and amended with dry top and root material from M. sativa, and in field trials on M. incognita and carrot cyst nematode Heterodera carotae with M. sativa pelleted meal. All amendments reduced root and soil population densities of target nematode species compared to non-treated and chemical controls, with a general improvement of plant growth and yield performances.

Molecular Characterization of Cyst Nematode Species (Heterodera spp.) from the Mediterranean Basin using RFLPs and Sequences of ITS‐rDNA

AbstractFifteen populations of cyst‐forming nematodes belonging to 11 known and one unidentified species collected in countries bordering the Mediterranean Sea were studied using polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP) and internal transcribed spacer (ITS)‐rDNA sequences. RFLP profiles generated by the restriction enzymes AluI, AvaI, Bsh1236I, HaeIII, Hin6I, MvaI, PstI and RsaI are presented for Heterodera carotae, H. ciceri, H. fici, H. filipjevi, H. goettingiana, H. hordecalis, H. humuli, H. mediterranea, H. ripae and H. schachtii. Molecular data support the first detection of H. filipjevi from wheat in Italy and H. ripae from nettle in Greece. A relative high level of sequence divergence between populations of H. hordecalis was observed. This suggests that two species might presently be grouped under this taxon. The phylogenetic relationship between the Mediterranean cyst‐forming nematode species is analysed based on the ITS‐rDNA sequences.

Effects of temperature and length of exposure on the mortality of the carrot cyst nematode, Heterodera carotae

A hatchability level of 72% of eggs in Heterodera carotae cysts was notsignificantly affected by keeping the cysts in water at up to 40 C for 2 h, but percentage hatch was strongly reduced by keeping the cysts for 2 h in water at 47.5, 50 and 52.5 C. Keeping soil with cysts of H. carotae with a hatchability before treatment of 27% at temperatures between 32.5 and 45 C for 2 and 4 h did not affect percentage hatch of the eggs in these cysts but longer exposures increased it in two ways and decreased it in one way. This resulted in a positive correlation between percentage hatch and length of exposure up to a smaller maximum hatch the higher the temperature. At still longer exposures percentage hatch was negatively correlated with length of exposure. Whether the decrease is due to mortality of the eggs or, partly, to reversible inactivation remains to be investigated. In contrast to hatching tests with root diffusate, probably almost all eggs will hatch in the field under a host crop. Therefore, only the adverse effect of the heat treatments on hatching is ofpracticalimportance for the estimation of the effect of solarization. To separate it from the effects that increased percentage hatch in the tests, a model was made of the interaction of the separate effects. It was based on linear relationships between log duration of exposure to different temperatures and probit relative increase and decrease of hatching rates, and linear relationships between treatment temperature and log length of exposure required to obtain a given effect of one of the two increasing effects and of the decreasing effect of the treatments. The coefficient of variation of hatchabilities that were affected by the increasing but not by the decreasing effect of the treatments, relative to those predicted by the model, was 7.1% against 7.8% after treatments that had no effect on percentage hatch, whereas the relationship between log length of exposure and probit percent reduction of hatchability by the treatments was linear. The relationship between probit hatchability ( h ) due to the decreasing affect of the treatments and temperature ( T ) during t hours is probit h = a b log t dTr - T in which T is a reference temperature. For T - 10 T r r T = 45 C, a = 11.047, b = 1.44 and d = 0.637 for H. carotae . A good r T r approximation of this relationship is log h= k l t dTr - T with k = 2.453, - T T r l = 0.03735 and d = 0.637. These relationships probably also apply, with T r other parameter values, to other nematode species. To calculate the effect of fluctuating temperatures t dTr - T is replaced in the equations by T Sigma (t dTr - T). T

The Biology of Heterodera Carotae

The biology of Heterodera carotae was investigated under laboratory and field conditions in Italy. Juveniles emerged from cysts all the year round and in greatest numbers (37.6-40.6%) at 15-20°C. Egg hatch was also substantial at 5-10°C, but negligible at 25°C. The age of carrot plants affected hatch and most juveniles (67-80.4%) emerged in root leachate from 5-7 week old carrots. Forty six per cent of the eggs from the egg masses were still viable at the end of the summer, but in November only 7 % of the eggs in soil were eggs from egg masses. Invasion of carrot roots by juveniles occurred over a range of temperatures from 8 to 20°C, but no nematode development was observed at 8°C. Heterodera carotae required 26 and 36 days to develop females and cysts at 20°C, when 260 and 360 day degrees above 10°C, respectively, had accumulated. Second stage juveniles appeared within egg masses and cysts after an accumulation of 510 and 570 day degrees above 10°C, respectively. Two generations can be completed by the nematode during a carrot growing season and no nematodes invade carrot roots in summer.

Hatching of Heterodera Carotae and H. Avenae

Hatching tests were done in Southern Italy using cysts of Heterodera carotae and H. avenae, and egg sacs of H. carotae. Juveniles hatched from eggs in newly formed cysts of the first species after 2 months whereas eggs in H. avenae cysts and H. carotae eggs sacs hatched promptly. High summer temperatures inhibited hatching but it commenced again in autumn, when the temperature dropped. Emergence from cysts of H. carotae was stimulated only by host plant root diffusate when incubated at a suitable temperature.