Plant Nematode Species Research Articles

Rapid diagnosis of Ditylenchus destructor by loop-mediated isothermal amplification assay based on 28S rRNA sequences

Ditylenchus destructor is an important plant pathogenic nematode in agricultural production and is listed as a quarantine pest by many countries and regions. Traditional identification depending on morphological characteristics requires taxonomic expertise and is difficult to apply for rapid identification of plant nematode species. Currently, with the development of molecular biology, many novel tools have been explored for the rapid identification of plant nematodes. In this study, loop-mediated isothermal amplification (LAMP) was used to conduct rapid detection of D. destructor from various origins. First, a set of new LAMP specific primers (DdBIP, DdFIP, DdF3, DdB3 and DdLF) were designed based on 28S rRNA sequences of D. destructor, and LAMP reaction conditions were optimized to use concentrations of 6.0 mmol L−1 Mg2+, 1.0 mmol L−1 dNTPs, and 0.2 U μL−1 Bst 2.0 DNA polymerase, performed at 65 °C for 50 min. Second, the specificity, stability and sensitivity of this method were confirmed using DNA from a single egg and juvenile, male and female nematodes of D. destructor and from D. destructor mixed with various nematode species and soil and tissue samples. The amplification products were detected by checking the ladder-like bands using electrophoresis and visualizing mixture colour changes using SYBR Green I dye. The results showed that the LAMP method could not only detect and identify a single egg and juvenile, female and male nematodes and different populations from different hosts and geographical origins of D. destructor but also directly detect D. destructor from mixed samples of various nematode species, plant tissue and soil. The sensitivity of LAMP amplification was a 1000-fold dilution of the template DNA from an individual female nematode. This report is the first to use LAMP to directly detect D. destructor from samples mixed with various nematode species and with plant tissue and soil.

Morphological and molecular characterization of Trophurus wuhuensis n. sp. (Nematoda: Telotylenchinae) from soil associated with Cinnamomum camphora in China.

A new plant nematode species, Trophurus wuhuensis n. sp., was collected from the soil associated with Cinnamomum camphora in Wuhu, Anhui Province, China. The new species is characterized by having a female with a slender body 660.5-801.5 μm in length, stylet 12-14 μm long, knobs directed laterad, lateral field marked by short and scattered grooves, post-vulval uterine sac shorter than vulval body diameter, post-rectal intestinal sac absent, tail cylindroid, terminus with deep wrinkles; and male with a pointed tail terminus and spicules 16-18 μm long. The internal transcribed spacer sequences of ribosomal DNA (ITS rDNA) and partial 18S ribosomal DNA (18S rDNA) from T. wuhuensis n. sp. were amplified and sequenced. A phylogenetic analysis based on sequences of 18S rDNA fragments is given in this study.

Morphology and molecular analysis of Paratylenchus guangzhouensis n. sp. (Nematoda: Paratylenchinae) from the soil associated with Bambusa multiplex in China

A new plant nematode species named Paratylenchus guangzhouensis n. sp. was extracted from the soil associated with Bambusa multiplex in Guangzhou, Guangdong Province, China. The new species is characterized by having the female with a small body 217–247 μm long, a slender stylet 43–49.5 μm long, head rounded without distinct submedian lobes, amphid openings large, and bow-tie like in shape, oral opening with two prominent ridges laterally, lateral field with three distinct lines, small lateral vulval flaps and tail tip finely rounded to bluntly rounded; male with a 237.5 μm long body, spicules 15.5 μm long, stylet lacking and pharynx degenerate; and the fourth-stage juvenile with a stylet. The internal transcribed spacer sequences of ribosomal RNA (ITS rRNA) gene of the new species were amplified and sequenced, and a phylogenetic tree inferred from the ITS rRNA gene is given in this study.

Morphological and molecular characterisation of Hemicaloosia guangzhouensis n. sp. (Nematoda: Caloosiidae) from China

A new plant nematode species, Hemicaloosia guangzhouensis n. sp., collected from the soil associated with Schima superba in a forest park in Guangzhou, China is described. The new species is characterized by having a female with a slender body, 798–961 μm in length, stylet 59–69 μm long, tail 115–145 μm long, a membranous sheath closely adpressed to the entire body, lateral fields marked by one longitudinal line with breaks and anastomoses of transverse striae, total number of annuli 282–313, two lip annuli both projecting slightly anterior, excretory pore 51–58 annuli from anterior end, the number of annulus between vulva and anus 7–11, the number of annulus from vulva to tail terminus 58–69, spermatheca empty; and male unknown. The internal transcribed spacer of ribosomal RNA (ITS rRNA) gene, D2-D3 region of 28S ribosomal RNA (28S rRNA) gene, partial 18S ribosomal RNA (18S rRNA) gene of the new species were amplified and sequenced. The 50 % majority rule consensus trees inferred from ITS rRNA, 28S rRNA, partial 18S rRNA gene sequences of H. guangzhouensis n. sp. and some other species in the suborder Criconematina are also presented in this study. PCR-ITS-RFLP diagnostic profile generated by six restriction enzymes for H. guangzhouensis n. sp. is given.

Online appendix 1:Plant nematodes in South Africa. 12. Checklist of plant nematodes of the protected areas of the Eastern Cape Province

Soil-inhabiting nematodes, including plant-parasitic nematodes, are considered to be the most abundant multicellular organisms in the soil, and of particular interest since they are an integral part of the interlocking chain of nutrient conversions. Because of their abundance and relative susceptibility to both physical and chemical changes, these organisms are used as indicator organisms. The National Collection of Nematodes (NCN) consists of a core collection, the Meloidogyne Collection and the Juan Heyns Collection, which are housed at the Plant Protection Research Institute of the Agricultural Research Council in Pretoria. Vast amounts of biodiversity data are contained in NCN, and the digitising of the collection from 2007 to 2014 yielded unpublished locality information, especially datasets of plant nematodes reported from protected areas of the Eastern Cape. Two hundred and thirty plant nematode species belonging to 36 genera were reported from the Eastern Cape. Of these, only 80 were from protected areas, whilst 163 were from uncultivated areas (outside protected areas) and 148 from cultivated areas. Ten species were described from protected areas, namely Criconemoides silvicola, Meloinema silvicola, Ogma tuberculatum, Paralongidorus cebensis, Paralongidorus hanliae, Scutellonema tsitsikamense, Trichodorus vandenbergae, Xiphinema erriae, Xiphinema ornatizulu and Xiphinema simplex. Only M. silvicola, O. tuberculatum, P. cebensis and S. tsitsikamense were not reported from other provinces, suggesting endemism.Conservation implications: The diversity of nematode fauna is not adequately protected as most nematode biodiversity in the Eastern Cape lies outside protected areas, with only 80 of the 230 plant-feeding nematode species in the province being reported from protected areas.

Plant nematodes in South Africa. 12. Checklist of plant nematodes of the protected areas of the Eastern Cape Province

Soil-inhabiting nematodes, including plant-parasitic nematodes, are considered to be the most abundant multicellular organisms in the soil, and of particular interest since they are an integral part of the interlocking chain of nutrient conversions. Because of their abundance and relative susceptibility to both physical and chemical changes, these organisms are used as indicator organisms. The National Collection of Nematodes (NCN) consists of a core collection, the Meloidogyne Collection and the Juan Heyns Collection, which are housed at the Plant Protection Research Institute of the Agricultural Research Council in Pretoria. Vast amounts of biodiversity data are contained in NCN, and the digitising of the collection from 2007 to 2014 yielded unpublished locality information, especially datasets of plant nematodes reported from protected areas of the Eastern Cape. Two hundred and thirty plant nematode species belonging to 36 genera were reported from the Eastern Cape. Of these, only 80 were from protected areas, whilst 163 were from uncultivated areas (outside protected areas) and 148 from cultivated areas. Ten species were described from protected areas, namely Criconemoides silvicola, Meloinema silvicola, Ogma tuberculatum, Paralongidorus cebensis, Paralongidorus hanliae, Scutellonema tsitsikamense, Trichodorus vandenbergae, Xiphinema erriae, Xiphinema ornatizulu and Xiphinema simplex. Only M. silvicola, O. tuberculatum, P. cebensis and S. tsitsikamense were not reported from other provinces, suggesting endemism.Conservation implications: The diversity of nematode fauna is not adequately protected as most nematode biodiversity in the Eastern Cape lies outside protected areas, with only 80 of the 230 plant-feeding nematode species in the province being reported from protected areas.

Plant nematodes in South Africa. 11. Checklist of plant nematodes of the protected areas of KwaZulu-Natal

Nematodes are some of the most abundant soil organisms and are an essential part of soil ecology. These organisms are used as indicator organisms and can be linked to soil health. Biological collections contain vast amounts of data, with the National Collection of Nematodes housed at the Plant Protection Research Institute, Agricultural Research Council being no different. During the digitising of the collection a number of unpublished records of plant nematodes reported from protected areas in KwaZulu-Natal were found in the South African Plant-Parasitic Nematode Survey database. A total of 222 plant nematode species belonging to 39 genera were reported from the province, with only 94 of these species reported from the protected areas and 172 and 159 species reported from uncultivated (outside the protected areas) and cultivated areas, respectively. Only nine species, Criconema silvum, Criconema talanum, Helicotylenchus marethae, Ogma dracomontanum, Ogma louisi, Ogma ueckermanni, Paralongidorus deborae, Trichodorus rinae and Xiphinemella marindae were described from protected areas, whilst O. dracomontanum, P. deborae and T. rinae were subsequently also reported from other provinces. Conservation implications: A higher degree of diversity of nematodes was observed in the unprotected areas of the province. The observation suggests that nematode fauna, and by implication also other invertebrates, are not adequately protected.

Online Appendix: Plant nematodes in South Africa. 11. Checklist of plant nematodes of the protected areas of KwaZulu-Natal

Nematodes are some of the most abundant soil organisms and are an essential part of soil ecology. These organisms are used as indicator organisms and can be linked to soil health. Biological collections contain vast amounts of data, with the National Collection of Nematodes housed at the Plant Protection Research Institute, Agricultural Research Council being no different. During the digitising of the collection a number of unpublished records of plant nematodes reported from protected areas in KwaZulu-Natal were found in the South African Plant-Parasitic Nematode Survey database. A total of 222 plant nematode species belonging to 39 genera were reported from the province, with only 94 of these species reported from the protected areas and 172 and 159 species reported from uncultivated (outside the protected areas) and cultivated areas, respectively. Only nine species, Criconema silvum, Criconema talanum, Helicotylenchus marethae, Ogma dracomontanum, Ogma louisi, Ogma ueckermanni, Paralongidorus deborae, Trichodorus rinae and Xiphinemella marindae were described from protected areas, whilst O. dracomontanum, P. deborae and T. rinae were subsequently also reported from other provinces. Conservation implications: A higher degree of diversity of nematodes was observed in the unprotected areas of the province. The observation suggests that nematode fauna, and by implication also other invertebrates, are not adequately protected.

Immunolocalization of a putative cuticular collagen protein in several developmental stages of Meloidogyne arenaria, Globodera pallida and G. rostochiensis.

The monoclonal antibody IACR-CCNj.3d has previously been used to isolate a gene (gp-col-8) with strong similarity to cuticular collagen from a mixed stage Globodera pallida cDNA expression library. The antibody has also been shown to label specifically the amphidial canal of pre-parasitic second stage juveniles (J2) of several plant nematode species without any reactivity on the cuticular surface, indicating that this protein is either not present or is inaccessible on the cuticular surface. This paper investigates the cross-reactivity of Mab IACR-CCNj.3d with Meloidogyne arenaria and the localization of the putative collagen protein on the cuticular surface of parasitic stages in planta and on the cuticular surface of juveniles inside eggs. The antigen was shown to be present in all developmental stages of the two species of potato cyst nematodes and M. arenaria. The antibody bound strongly to the amphidial canal and hypodermis of pre-parasitic J2 and adult females. The antigen was present on the cuticular surface of the sausage-shaped J2 in planta and of first stage juveniles (J1) inside the eggs. The presence of collagen on the surface of the cuticle of moulting stages of plant parasitic nematodes has been observed for the first time. It is clear that this protein has a role in the construction of the cuticle of the first stage juveniles and parasitic second stage juveniles, during moulting inside the eggs and in the root tissue, respectively.

Effects of intact glucosinolates and products produced from glucosinolates in myrosinase-catalyzed hydrolysis on the potato cyst nematode (Globodera rostochiensis Cv. Woll).

The potato cyst nematode (Globodera rostochiensis cv. Woll) is responsible for large yield losses in the potato crop, and opportunities for reducing the attack of these plant nematode species are, therefore, important. This study has been devoted to the testing of the in vitro effects on the potato cyst nematode of eight glucosinolates [prop-2-enyl-, but-3-enyl-, (R)-4-methylsulfinylbut-3-enyl-, benzyl-, phenethyl-, 4-hydroxybenzyl-, (2S)-2-hydroxybut-3-enyl-, and (2R)-2-hydroxy-2-phenylethylglucosinolate] as well as the effects of the products of this myrosinase-catalyzed hydrolysis. The glucosinolates were used at three concentrations, 0.05, 0.3, and 1.0 mg/mL, in the presence or absence of the enzyme myrosinase. The effects of the compounds on the mortality were monitored every 8 h for a 72 h period. No effects were found for any of the intact glucosinolates. However, when active myrosinase was included with 1 mg/mL phenethylglucosinolate at pH 6.5, 100% mortality was observed within just 16 h. A similar effect was achieved at the same concentration of benzyl- and prop-2-enylglucosinolates in the myrosinase-containing solutions, although longer exposures were required (24 and 40 h, respectively). The main aglucone products released from the glucosinolates with pronounced effects on the nematodes were shown to be the corresponding isothiocyanates. The results suggest that mixtures of these specific glucosinolates and active myrosinase or autolysis of plant materials containing these enzymes and glucosinolates might be used to control the potato cyst nematode in the soil.

The differentiation of parasitic nematodes using random amplified polymorphic DNA.

DNA from species and races of plant parasitic nematodes (Meloidogyne, Globodera and Heterodera) and a human parasitic nematode (Trichinella) were subjected to polymerase chain reaction amplification using one arbitrary primer (M-10). This technique results in relatively simple DNA profiles that include polymorphic markers known as random amplified polymorphic DNA (RAPDs). The RAPD profiles of the plant nematode species of Meloidogyne made possible the identification of M. incognita and M. hapla, but no differences were found between the patterns of M. javanica, M. arenaria and M. graminicola. Moreover, the four races of M. incognita were indistinguishable by this primer. In contrast, when races of the plant nematode Globodera rostochiensis (Ro1 and Ro2/3) were studied under the same RAPDs conditions, a race specific profile allows these two most devastating races to be differentiated. When DNAs of eight Trichinella isolates were subjected to RAPD studies, four different patterns were identified, corresponding to the four Trichinella clusters previously defined by isozyme polymorphism.

Occurrence of Plant Parasitic Nematodes in Punjab, India

Abstract Forty-seven plant nematode species were found associated with 33 crops, bringing the total number of plant nematode species reported from the state to 68. Meloidogyne incognita, M. arenaria, Pratylenchus zeae, Tylenchorhynchus brassicae, Tylenchulus semipenetrans, Rotylenchulus reniformis, Xiphincma americanum, Heterodera avenae and H. zeae cause extensive damage. Vegetables were the most adversely affected of all the crops investigated.