Wen-Jia Wu

Cathepsin B Cysteine Proteinase is Essential for the Development and Pathogenesis of the Plant Parasitic Nematode Radopholus similis

Radopholus similis is an important plant parasitic nematode which severely harms many crops. Cathepsin B is present in a wide variety of organisms, and plays an important role in many parasites. Understanding cathepsin B of R. similis would allow us to find new targets and approaches for its control. In this study, we found that Rs-cb-1 mRNA was expressed in esophageal glands, intestines and gonads of females, testes of males, juveniles and eggs in R. similis. Rs-cb-1 expression was the highest in females, followed by juveniles and eggs, and was the lowest in males. The maximal enzyme activity of Rs-CB-1 was detected at pH 6.0 and 40 °C. Silencing of Rs-cb-1 using in vitro RNAi (Soaking with dsRNA in vitro) not only significantly inhibited the development and hatching of R. similis, but also greatly reduced its pathogenicity. Using in planta RNAi, we confirmed that Rs-cb-1 expression in nematodes were significantly suppressed and the resistance to R. similis was significantly improved in T2 generation transgenic tobacco plants expressing Rs-cb-1 dsRNA. The genetic effects of in planta RNAi-induced gene silencing could be maintained in the absence of dsRNA for at least two generations before being lost, which was not the case for the effects induced by in vitro RNAi. Overall, our results first indicate that Rs-cb-1 plays key roles in the development, hatching and pathogenesis of R. similis, and that in planta RNAi is an effective tool in studying gene function and genetic engineering of plant resistance to migratory plant parasitic nematodes.

Pin Nematode Slow Decline of Anthurium andraeanum, a New Disease Caused by the Pin Nematode Paratylenchus shenzhenensis

High population densities of the plant-parasitic nematode Paratylenchus shenzhenensis have been observed in association with stunted anthurium in Shenzhen, Guangdong Province, China. P. shenzhenensis is now recognized as a new pathogen that causes severe damage to anthurium. Commercial fields of anthurium infected by P. shenzhenensis present a patchy distribution of plants that are usually stunted and in decline and have yellowing leaves. Roots that are heavily infected by this nematode present brown, dark-brown, or black rot, and the entire root mass is often destroyed. In this study, a method of culturing P. shenzhenensis on carrot disks was established, and the effects of temperature and time on the reproduction rate (Rr = final number of nematodes/initial number of nematodes) of this nematode were assessed. The optimum temperature for culturing P. shenzhenensis on carrot disks is 25°C and, after inoculation with 20 females and 10 males at 25°C for 10 weeks, Rr reached 3,834. At the optimum temperature, a suitable extension culture time or an increase in initial inoculum density will improve the reproductive efficiency of P. shenzhenensis. Kochs postulates were fulfilled by inoculating P. shenzhenensis on the roots of anthurium and demonstrating the pathogenicity of the nematode. Histological analyses showed that P. shenzhenensis feed endoparasitically on the roots of anthurium; whole nematode bodies were observed in the outermost epidermal cells and root hairs, and the cell walls and middle lamellae were partially dissolved because of nematode migration and feeding.

Morphology and molecular analysis of Paratylenchus nanjingensis n. sp. (Nematoda: Paratylenchinae) from the rhizosphere soil of Pinus massoniana in China

Paratylenchus nanjingensis n. sp. was obtained from Nanjing, Jiangsu Province, China. This new species is characterized by having a female with a slender, vermiform body (243-279 μm), head with distinct submedian lobes, slender and long stylet (64-68 μm), anchor-shaped stylet knobs, excretory pore anterior to the level of the stylet knobs, small lateral vulval flaps and lateral field with four lines; and male with more distinct body annuli, stylet lacking and pharynx degenerate. The internal transcribed spacer sequences of ribosomal RNA (ITS rRNA) gene of the new species were amplified and sequenced in this study. The phylogenetic relationships of the new species with other Paratylenchus species using the ITS rRNA gene sequences are given.

Description and molecular analysis of Tylencholaimus helanensis sp. n. from China (Dorylaimida, Tylencholaimidea)

Abstract A new species, Tylencholaimushelanensissp. n., extracted from the rhizosphere soil of unidentified grasses from Helan Mountain, Inner Mongolia, China was identified. The new species is characterized by having a body length of 0.93–1.07 mm with the lip region approximately one-quarter of the body diameter at the posterior end of the neck region wide; female didelphic-amphidelphic; pars proximalis vaginae violin-shaped. Males were not found. SEM observations of the new species were made and a phylogenetic analysis of both the 18S rDNA and the D2-D3 region of 28S rDNA is presented.

Nematicidal protease genes screened from a soil metagenomic library to control Radopholus similis mediated by Pseudomonas fluorescens pf36

Controlling Radopholus similis, an important phytopathogenic nematode, is a challenge worldwide. Herein, we constructed a metagenomic fosmid library from the rhizosphere soil of banana plants, and six clones with protease activity were obtained by functionally screening the library. Furthermore, subclones were constructed using the six clones, and three protease genes with nematicidal activity were identified: pase1, pase4, and pase6. The pase4 gene was successfully cloned and expressed, demonstrating that the protease PASE4 could effectively degrade R. similis tissues and result in nematode death. Additionally, we isolated a predominant R. similis-associated bacterium, Pseudomonas fluorescens (pf36), from 10 R. similis populations with different hosts. The pase4 gene was successfully introduced into the pf36 strain by vector transformation and conjugative transposition, and two genetically modified strains were obtained: p4MCS-pf36 and p4Tn5-pf36. p4MCS-pf36 had significantly higher protease expression and nematicidal activity (p < 0.05) than p4Tn5-pf36 in a microtiter plate assay, whereas p4Tn5-pf36 was superior to p4MCS-pf36 in terms of genetic stability and controlling R. similis in growth pot tests. This study confirmed that R. similis is inhibited by the associated bacterium pf36-mediated expression of nematicidal proteases. Herein, a novel approach is provided for the study and development of efficient, environmentally friendly, and sustainable biocontrol techniques against phytonematodes.

A new species of the genus Discolaimus Cobb, 1913 (Nematoda: Dorylaimida: Qudsianematidae) from Qinghai, China.

A new species of the family Qudsianematidae Jairajpuri, 1965 collected from soil from Qinghai Province, China is described as Discolaimus anemaqen n. sp. It is mainly characterized by having a disc-like lip region with six labial sectors separated by six Y-shaped radial grooves; amphid fovea an inverted-stirrup shape; odontostyle aperture 53-59% of its length; anterior part of pharynx with two swellings, basal part expanded gradually with a sheath of tissue visible ventrally and at the base; no groups of large cells observed in cardiac region; female genital system amphidelphic; tail dorsally convex, conoid, with flat ventral side and bluntly rounded terminus, and males not found. The new species is close to D. major Thorne, 1939, D. similis Thorne, 1939 and D. silvicolus Sauer & Annells, 1985 in most measurements, but can be differentiated from them by lip morphology, pharynx structure, body size, and tail shape. A key to the species of Discolaimus is also provided.

Arabidopsis thaliana as a suitable model host for research on interactions between plant and foliar nematodes, parasites of plant shoot

The rice white tip nematode (RWTN), Aphelenchoides besseyi and the chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi are migratory plant parasitic nematodes that infect the aboveground parts of plants. In this research, Arabidopsis thaliana was infected by RWTN and CFN under indoor aseptic cultivation, and the nematodes caused recognizable symptoms in the leaves. Furthermore, RWTN and CFN completed their life cycles and proliferated. Therefore, A. thaliana was identified as a new host of RWTN and CFN. The optimum inoculum concentration for RWTN and CFN was 100 nematodes/plantlet, and the optimum inoculum times were 21 and 24 days, respectively. For different RWTN populations, the pathogenicity and reproduction rates were different in the A. thaliana Col-0 ecotype and were positively correlated. The optimum A. thaliana ecotypes were Col-0 and WS, which were the most susceptible to RWTN and CFN, respectively. Additionally, RWTN was ectoparasitic and CFN was ecto- and endoparasitic in A. thaliana. The RWTN and CFN migrated from inoculated leaves to the entire plantlet, and the number of nematodes in different parts of A. thaliana was not correlated with distance from the inoculum point. This is a detailed study of the behavior and infection process of foliar nematodes on A. thaliana.

Morphological and karyotypic differences within and among populations of Radopholussimilis.

Abstract Twenty populations of Radopholus similis from three countries and different hosts (19 populations from ornamental plants and one population from ginger) were compared using morphological characters, morphometrics and karyotype between progeny from both single females and 30 females of each population. Morphological diversity existed in and among the populations, even within the progeny nematodes from single nematodes compared to that of 30 females. The labial disc shape, the number of head annuli, the terminated position of lateral lips, the number of genital papillae before cloacal apertures and female and male tail terminal shape showed variation. In addition, genital papillae arranged in a double row before cloacal apertures was first found in two ornamental populations. The karyotype of all the 20 populations was n = 5. Combining our results and previous studies, we support that Radopholus citrophilus is a synonym of Radopholus similis, and that it is not possible to distinguish physiological races or pathotypes of Radopholus similis according to morphological characters or karyotype.