Population Dynamics of Meloidogyne arenaria and Pasteuria penetrans in a Long-Term Crop Rotation Study

The endospore-forming bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.). The primary objective of this study was to determine the effect of crop sequence on abundance of P. penetrans. The experiment was conducted from 2000 to 2008 at a field site naturally infested with both the bacterium and its host Meloidogyne arenaria and included the following crop sequences: continuous peanut (Arachis hypogaea) (P-P-P) and peanut rotated with either 2 years of corn (Zea mays) (C-C-P), 1 year each of cotton (Gossypium hirsutum) and corn (Ct-C-P), or 1 year each of corn and a vegetable (V-C-P). The vegetable was a double crop of sweet corn and eggplant (Solanum melongena). A bioassay with second-stage juveniles (J2) of M. arenaria from a greenhouse (GH) population was used to estimate endospore abundance under the different crop sequences. A greater numerical increase in endospore densities was expected in the P-P-P and V-C-P sequences than in the other sequences because both peanut and eggplant are good hosts for M. arenaria. However, endospore densities, as determined by bioassay, did not substantially increase in any of the sequences during the 9-year experiment. To determine whether the nematode population had developed resistance to the resident P. penetrans, five single egg-mass (SEM) lines from the field population of M. arenaria were tested alongside the GH population for acquisition of endospores from the field soil. Four of the five SEM lines acquired 9 to 14 spores/J2 whereas the GH population and one of the SEM lines acquired 3.5 and 1.8 spores/J2, respectively. Endospore densities estimated with the four receptive SEM lines were highest in the P-P-P plots (14-20 spores/J2), intermediate in the V-C-P plots (6-7 spores/J2), and lowest in the Ct-C-P plots (< 1 spore/J2). These results indicate that the field population of M. arenaria is heterogeneous for attachment of P. penetrans endospores. Moreover, spore densities increased under intensive cropping of hosts for M. arenaria, but the GH population of the nematode was not receptive to spore attachment. However, previously, the GH population was very receptive to spore acquisition from this field site. One explanation for this inconsistency is that the M. arenaria population in the field became resistant to the dominant subpopulation of P. penetrans that had been present, and this led to the selection of a different subpopulation of the bacterium that is incompatible with the GH population.     

Enhancement of Cylindrocladium crotalariae Root Rot by Meloidogyne arenaria (Race 2) on a Peanut Cultivar Resistant to Both Pathogens

Two populations of Meloidogyne arenaria (race 2, incompatible on peanut) enhanced development of Cylindrocladium black rot (CBR) on CBR-resistant peanut cv. NC 3033 in greenhouse factorial experiments. Nematode populations 256 and 486 (0, 10³, 10⁴ eggs per 15-cm pot) were tested in all combinations with Cylindrocladium crotalariae (0, 0.5, 5, 50 microsclerotia per cm³ of soil). Root-rot index increased in the presence of either population. Positions but not slope values of inoculum density-disease curves were changed by both populations, indicating increased efficiency of microsclerotia when peanuts were grown in the presence of these nematodes. Although little or no reproduction occurred with either nematode population on NC 3033, larvae of 256 and 486 penetrated roots. Meloidogyne arenaria 486 did not induce root galls and was not snccessful in establishing feeding sites. Meloidogyne arenaria 256 produced a few very small eliptical galls and had a range of success in establishing a feeding site, varying from no giant cell development to large giant cell with production of a few eggs.     

Nematicidal activity and chemical component of Poria cocos

Poria cocos, a famous traditional Chinese medicine, was found to have nematicidal activity in experiments searching for nematicidal fungi. The experiment showed it could kill 94.9% of the saprophytic nematode, Panagrellus redivivue, 92.6% of the root-knot nematode, Meloidogyne arenaria, and 93.5% of the pine nematode, Bursaphelenchus xylophilus, on PDA plate within 12 hours. According to the nematicidal activity, three new compounds, 2, 4, 6-triacetylenic octane diacid, 2, 4, 5, 6-tetrahydroxyhexanoic acid and 3, 4-dihydroxy-2-keto-n-butyl 2,4,5,6-tetrahydroxyhexanate, were isolated from submerged cultures of Poria cocos. Of these, 2, 4, 6-triacetylenic octane diacid could kill 83.9% Meloidogyne arenaria and 73.4% Panagrellus redivivus at 500 ppm within 12 hours. Here, it is reported for the first time that Poria cocos has nematicidal activity.     

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.     

Protein variability in Meloidogyne spp. (Nematoda:Meloidogynidae) revealed by two-dimensional gel electrophoresis and mass spectrometry

Total protein variation as revealed by two-dimensional electrophoresis (2D-E) was studied in 18 isolates from populations of Meloidogyne arenaria (six isolates), Meloidogyne incognita (10 isolates), and Meloidogyne javanica (one isolate) plus an unclassified isolate. Gels (80 x 60 x 0.75 mm) were silverstained and digitized in order to compare their protein patterns. Optical density and position of protein patterns were measured using statistical cluster analysis and computer-assisted image analysis software. Only those protein stains or positions that were clearly defined (i.e., without background) were considered. The number of positions in gels ranged from 86 to 203. Each of these positions had 95 clearly expressed proteins that were present in at least two replicates for each isolate. Spot position was considered a taxonomical character with two different states: presence (1) and absence (0). Accordingly, genetic distance was estimated among isolates and species, and a phylogenetic tree was constructed following the cladistic approach based on maximum parsimony analysis. Isolates of M. arenaria--M. javanica--Meloidogyne sp. and of M. incognita formed two separate monophyletic groups. Both groups were clearly defined on the basis of two sets of protein positions that can be considered as diagnostic characters. An attempt to identify these proteins by mass spectrometry was made. Group diagnostic proteins for M. incognita and M. arenaria (and for other proteins common to all isolates) were distinguished by protonated mass signals in the MALDI fingerprinting spectrum.     

Distribution and Identification of Root-knot Nematodes from Turkey

Root knot nematodes are causing serious losses in protected cultivation fields in the West Mediterranean region of Turkey. Correct and confident identification of the plant parasitic nematodes is important for vegetable growing and breeding. Therefore, ninety-five populations of plant parasitic nematodes were collected from regional greenhouses. Previously described species-specific primers were used to identify Meloidogyne populations. The present study indicated that SEC-1F/SEC-1R and INCK14F-INCK14R primers for identifying of M. incognita, Fjav/Rjav and DJF/DJR primers for M. javanica and Far/Rar for M. arenaria primers can be effective tools to identify the Turkish root-knot nematode species. Dissemination ratios of the population were 64.2%, 28.4% and 7.3% for Meloidogyne incognita, M. javanica and M. arenaria, respectively. The results showed that M. incognita was the prominent root-knot nematode species in the West Mediterranean coastal areas of Turkey.     

Sensitive PCR Detection of Meloidogyne arenaria, M. incognita, and M. javanica Extracted from Soil

We have developed a simple PCR assay protocol for detection of the root-knot nematode (RKN) species Meloidogyne arenaria, M. incognita, and M. javanica extracted from soil. Nematodes are extracted from soil using Baermann funnels and centrifugal flotation. The nematode-containing fraction is then digested with proteinase K, and a PCR assay is carried out with primers specific for this group of RKN and with universal primers spanning the ITS of rRNA genes. The presence of RKN J2 can be detected among large numbers of other plant-parasitic and free-living nematodes. The procedure was tested with several soil types and crops from different locations and was found to be sensitive and accurate. Analysis of unknowns and spiked soil samples indicated that detection sensitivity was the same as or higher than by microscopic examination.     

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

SEQUENCE ANALYSIS OF A NOVEL INSECT PHOSPHOGLYCERATE MUTASE GENE FROM THE CHINESE HONEYBEE,APIS CERANA*

A clone inserted with 1 104 bp fragment containing a 765bp Open Reading Frame(ORF), encoding a putative 2,3‐bisphosphoglycerate(2,3BPG) dependent Phosphoglycerate mutase(dPGAM) that catalyzes the transfer of a phosphate group from the C3 carbon atom to the C2 carbon atom of phosphoglycerate, was screened by mass sequencing from the cDNA library of the venom glands of Apis cerana. The deduced amino acid sequence shared high similarities (39% ‐ 88%)with the dPGAM of 7 other organisms, but the similarities with the iPGAM of 4 other organisms were low (10% ‐ 12%). Moreover, the alignment of Ac‐PGAM with the dPGAMs from 7 other organisms showed that all the active site amino acid residues were conserved. This result shows that Ac‐PGAM is a typical dPGAM. Thus, this is the second PGAM gene reported in Insecta. Furthermore, phylogenetic analysis showed that the evolutionary tree of PGAMs reflects the systematic relationship of species.     

中华蜜蜂磷酸甘油变位酶（PhosphoglycerateMutase）基因的序列分析

在中华蜜蜂(Apis cerana)工蜂毒腺cDNA库内发现了一个插有1104bp外源片段的克隆,内含一个765bp的开放阅读框架(ORF),编码一个含有254．个氨基酸残基的依赖于2,3一二磷酸甘油酸的磷酸甘油变位酶(dPGAM),催化3一磷酸甘油和2一磷酸甘油之间的转化。推测的氨基酸序列与其他7种生物的dPGAM的相似性很高(39％-88％),而与其他4种不依赖于2,3-二磷酸甘油酸的磷酸甘油变位酶(iPGAM)的相似性则很低(10％-12％),氨基酸序列的多重联配表明组成dPGAM活性位点的氨基酸残基在包括中华蜜蜂在内的所有生物体内是十分保守的,Ac—PGAM是一种典型的dPGAM。这是昆虫纲中继在果蝇中发现PGAM基因后的第2个昆虫dPGAM基因,其对PGAM基因的结构与功能研究及对昆虫的分子生物学研究具有意义。同时,对PGAM的进化关系的分析表明该基因可以用作研究物种系统关系的一个依据。     

Rapid identification of cyst (Heterodera spp., Globodera spp.) and root-knot (Meloidogyne spp.) nematodes on the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) in cultures and field samples

Cyst and root-knot nematodes show high levels of gross morphological similarity. This presents difficulties for the study of their ecology in natural ecosystems. In this study, cyst and root-knot nematode species, as well as some ectoparasitic nematode species, were identified using the second internal transcribed spacer (ITS2) sequence variation detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP). The ITS2 region was sufficiently variable within the taxa investigated to allow species to be separated on the basis of minor sequence variation. The PCR primers used in this study were effective for 12 species with three genera within the Heteroderinae (Globodera pallida, G. rostochiensis, Heterodera arenaria/avenae, H. ciceri, H. daverti, H. hordecalis, H. mani, H. schachtii, H. trifolii, Meloidogyne ardenensis, M. duytsi and M. maritima). However, pathotypes of Globodera pallida and G. rostochiensis could not be distinguished. The method was tested at two coastal dune locations in The Netherlands (one in the lime-poor dunes of the north and one in calcareous dunes of the south) to determine the population structure of cyst nematodes. At each site, cyst nematodes were associated with three plant species: two plant species on the foredune (Elymus farctus and Ammophila arenaria) and one plant species occurring further inland (Calamagrostis epigejos). Two species of cyst nematodes, H. arenaria and H. hordecalis, were found. H. arenaria associated with vigorous A. arenaria and H. hordecalis in association with degenerating A. arenaria and C. epigejos. The field survey demonstrated that in coastal dunes abiotic factors may be the important affecting the distribution of cyst nematodes.     

Effects of the Mi-1 and the N root-knot nematode-resistance gene on infection and reproduction of Meloidogyne enterolobii on tomato and pepper cultivars

Meloidogyne enterolobii is widely considered to be an aggressive root-knot nematode species that is able to reproduce on root-knot nematode-resistant tomato and pepper cultivars. In greenhouse experiments, M. enterolobii isolates 1 and 2 from Switzerland were able to reproduce on tomato cultivars carrying the Mi-1 resistance gene as well as an N-carrying pepper cultivar. Reproduction factors (Rf) ranged between 12 and 109 depending on the plant cultivar, with M. enterolobii isolate 2 being more virulent when compared to isolate 1. In contrast, M. arenaria completely failed to reproduce on these resistant tomato and pepper cultivars. Although some variability in virulence and effectiveness of root-knot nematode-resistance genes was detected, none of the plant cultivars showed Rf values less than 1 or less than 10% of the reproduction observed on the susceptible cv. 'Moneymaker' (Rf = 23-44) used to characterize resistance. The ability of M. enterolobii to overcome the resistance of tomato and pepper carrying the Mi-1 and the N gene makes it difficult to manage this root-knot nematode species, particularly in organic farming systems where chemical control is not an option.     

Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species.

RAPD markers were used to characterize the genetic diversity and relationships of root-knot nematodes (RKN) (Meloidogyne spp.) in Brazil. A high level of infraspecific polymorphism was detected in Meloidogyne arenaria, Meloidogyne exigua, and Meloidogyne hapla compared with the other species tested. Phylogenetic analyses showed that M. hapla and M. exigua are more closely related to one another than they are to the other species, and illustrated the early divergence of these meiotically reproducing species from the mitotic ones. To develop a PCR-based assay to specifically identify RKN associated with coffee, three RAPD markers were further transformed into sequence-characterized amplified region (SCAR) markers specific for M. exigua, Meloigogyne incognita and Meloidogyne paranaensis, respectively. After PCR using the SCAR primers, the initial polymorphism was retained as the presence or absence of amplification. Moreover, multiplex PCR using the three pairs of SCAR primers in a single reaction enabled the unambiguous identification of each species, even in mixtures. Therefore, it is concluded that the method developed here has potential for application in routine diagnostic procedures.     

The heat-stable root-knot nematode resistance gene <Emphasis Type="Italic">Mi-9</Emphasis> from Lycopersicon peruvianum is localized on the short arm of chromosome 6

The tomato gene Mi-1 confers resistance to three species of root-knot nematodes, Meloidogyne spp. However, the resistance mediated by Mi-1 is inactive at soil temperatures above 28 degrees C. Previously, we identified and mapped a novel heat-stable nematode resistance gene from the wild species Lycopersicon peruvianum accession LA2157 on to chromosome 6. Here we report further characterization of this heat-stable resistance against three Mi-1-avirulent biotypes of Meloidogyne javanica, Meloidogyne arenaria and Meloidogyne incognita. Screening segregating F(2) and F(3) progenies, derived from an intraspecific cross between susceptible LA392 and resistant LA2157, for nematode resistance at 25 degrees C and 32 degrees C, revealed a simple dominant monogenic inheritance with all the biotypes tested. We designate this gene as Mi-9. As a first step towards cloning of Mi-9, we constructed a linkage map around this gene. A total of 216 F(2) progeny from the cross between LA392 and LA2157 were screened with M. javanica at 32 degrees C and with CT119 and Aps-1, markers that flank the genetic interval that contains the Mi-1 gene. DNA marker analysis indicated that these markers also flank Mi-9. Further mapping of recombinants with both RFLP and PCR-based markers localized Mi-9 to the short arm of chromosome 6 and within the same genetic interval that spans the Mi-1 region.     

Characterization of potentially functional 5S rRNA-encoding genes within ribosomal DNA repeats of the nematode Meloidogyne arenaria.

In the plant-parasitic nematode Meloidogyne arenaria, isolated rDNA repeats show length heterogeneity, and are unusual in that they contain putative 5S ribosomal RNA pseudogenes [Vahidi et al., J. Mol. Evol. 27 (1988) 222-227]. Potentially functional 5S rRNA-encoding genes can also be identified in various rDNA repeats, which appear to be tandemly organized in the genome.     

Preliminary study of the green algae chlorella (Chlorella vulgaris) for control on the root-knot nematode (Meloidogyne arenaria) in tomato plants and ectoparasite Xiphinema indexin grape seedlings

The alternative ecological methods require investigation of many organo-biological means for plant protection against dangerous root parasites such as root-knot nematode Meloidogyne arenaria and some ectoparasites (Xiphinema index). The Bulgarian organic product - dry extract of green alga Chlorella vulgaris ("The Golden Apple"-Plamen Barakov) is the latest product, which in comparative aspect gives the best results. Series of laboratory and pot experiments are carried out with tomato (cv. Bele and cv. Ideal) and grape seedlings (cv. Cabernet Sauvignon). Different dosages of Chlorella from 0.5 g to 2 g per plant/pot are investigated. The first results show that even low dosages had double effect - on the one hand they suppress the parasite development and on the other hand they strongly stimulate plant growing. The very important conclusion is that Chlorella vulgaris ignores the negative influence of M. arenaria and X. index. These results give us opportunity for future model and field investigations of Chlorella vulgaris with the aim of its practical application.