Congli Wang

Application of Pluronic gel to the study of root-knot nematode behaviour.

Pluronic F-127 is a stable, non-toxic, copolymer that forms a gel at room temperature and a liquid at 15°C when the concentration is 20-30%. Root-knot nematode (Meloidogyne spp.) second-stage juveniles can move freely through the gel and display attraction toward roots of tomato, Medicago truncatula, common bean and Arabidopsis. The excellent clarity of the gel allows examination of behavioural changes in the nematode as it reaches and infects its host. Attraction assays showed that Meloidogyne javanica and M. incognita move to roots much more rapidly than does M. hapla. Nematodes form aggregates when in contact with root tips suggesting that the presence of a signal from the nematode or from the root is involved in the attraction. Nematodes suspended in Pluronic gel without roots aggregate into balls after 1 to 2 days. A coverslip placed on the gel accelerates and serves as a focus for the aggregation, suggesting that lower oxygen, or perhaps a volatile attractant, is involved in this behaviour. These observations demonstrate that Pluronic gel is a useful medium for dissecting attraction of root-knot nematodes to their hosts and for studying additional aspects of their behaviour.

Ethylene Signaling Pathway Modulates Attractiveness of Host Roots to the Root-Knot Nematode Meloidogyne hapla

Infective juveniles of the root-knot nematode Meloidogyne hapla are attracted to the zone of elongation of roots where they invade the host but little is known about what directs the nematode to this region of the root. We found that Arabidopsis roots exposed to an ethylene (ET)-synthesis inhibitor attracted significantly more nematodes than control roots and that ET-overproducing mutants were less attractive. Arabidopsis seedlings with ET-insensitive mutations were generally more attractive whereas mutations resulting in constitutive signaling were less attractive. Roots of the ET-insensitive tomato mutant Never ripe (Nr) were also more attractive, indicating that ET signaling also modulated attraction of root-knot nematodes to this host. ET-insensitive mutants have longer roots due to reduced basipetal auxin transport. However, assessments of Arabidopsis mutants that differ in various aspects of the ET response suggest that components of the ET-signaling pathway directly affecting root length are not responsible for modulating root attractiveness and that other components of downstream signaling result in changes in levels of attractants or repellents for M. hapla. These signals may aid in directing this pathogen to an appropriate host and invasion site for completing its life cycle.

Determination of Preferred pH for Root-knot Nematode Aggregation Using Pluronic F-127 Gel

Root-knot nematodes (Meloidogyne spp.) are obligate endoparasites of a wide range of plant species. The infective stage is attracted strongly to and enters host roots at the zone of elongation, but the compounds responsible for this attraction have not been identified. We developed a simple assay to investigate nematode response to chemical gradients that uses Pluronic F-127, a synthetic block copolymer that, as a 23% aqueous solution, forms a liquid at low temperature and a gel at room temperature. Test chemicals are put into a modified pipette tip, or ‘chemical dispenser,’ and dispensers are inserted into the gel in which nematodes have been dispersed. Meloidogyne hapla is attracted to pH gradients formed by acetic acid and several other Brønsted acids and aggregates between pH 4.5 and 5.4. While this pH range was attractive to all tested root-knot nematode strains and species, the level of aggregation depended on the species/strain assessed. For actively growing roots, the pH at the root surface is most acidic at the zone of elongation. This observation is consistent with the idea that low pH is an attractant for nematodes. Root-knot nematodes have been reported to be attracted to carbon dioxide, but our experiments suggest that the observed attraction may be due to acidification of solutions by dissolved CO2 rather than to CO2 itself. These results suggest that Pluronic F-127 gel will be broadly applicable for examining responses of a range of organisms to chemical gradients or to each other.

QTL analysis for transgressive resistance to root-knot nematode in interspecific cotton (Gossypium spp.) progeny derived from susceptible parents.

The southern root-knot nematode (RKN, Meloidogyne incognita) is a major soil-inhabiting plant parasite that causes significant yield losses in cotton (Gossypium spp.). Progeny from crosses between cotton genotypes susceptible to RKN produced segregants in subsequent populations which were highly resistant to this parasite. A recombinant inbred line (RIL) population of 138 lines developed from a cross between Upland cotton TM-1 (G. hirsutum L.) and Pima 3–79 (G. barbadense L.), both susceptible to RKN, was used to identify quantitative trait loci (QTLs) determining responses to RKN in greenhouse infection assays with simple sequence repeat (SSR) markers. Compared to both parents, 53.6% and 52.1% of RILs showed less (P<0.05) root-galling index (GI) and had lower (P<0.05) nematode egg production (eggs per gram root, EGR). Highly resistant lines (transgressive segregants) were identified in this RIL population for GI and/or EGR in two greenhouse experiments. QTLs were identified using the single-marker analysis nonparametric mapping Kruskal-Wallis test. Four major QTLs located on chromosomes 3, 4, 11, and 17 were identified to account for 8.0 to 12.3% of the phenotypic variance (R2) in root-galling. Two major QTLs accounting for 9.7% and 10.6% of EGR variance were identified on chromosomes 14 and 23 (P<0.005), respectively. In addition, 19 putative QTLs (P<0.05) accounted for 4.5–7.7% of phenotypic variance (R2) in GI, and 15 QTLs accounted for 4.2–7.3% of phenotypic variance in EGR. In lines with alleles positive for resistance contributed by both parents in combinations of two to four QTLs, dramatic reductions of >50% in both GI and EGR were observed. The transgressive segregants with epistatic effects derived from susceptible parents indicate that high levels of nematode resistance in cotton may be attained by pyramiding positive alleles using a QTL mapping approach.

Root-knot nematodes exhibit strain-specific clumping behavior that is inherited as a simple genetic trait.

Root-knot nematodes are obligate parasites of a wide range of plant species and can feed only on the cytoplasm of living plant cells. In the absence of a suitable plant host, infective juveniles of strain VW9 of the Northern root-knot nematode, Meloidogyne hapla, when dispersed in Pluronic F-127 gel, aggregate into tight, spherical clumps containing thousands of worms. Aggregation or clumping behavior has been observed in diverse genera in the phylum Nematoda spanning free-living species such as Caenorhabditis elegans as well as both plant and animal parasites. Clumping behavior differs between strains of M. hapla and occurs with other species within this genus where strain-specific differences in clumping ability are also apparent. Exposure of M. hapla juveniles to a gradient formed using low levels of cyanide promotes formation of clumps at a preferred cyanide level. Analysis of F2 lines from a cross of M. hapla strains that differ in clump-forming behavior reveals that the behavior segregates as a single, major locus that can be positioned on the genetic map of this nematode. Clumping behavior may be a survival strategy whose importance and function depend on the niche of the nematode strain or species.

Ethylene response pathway modulates attractiveness of plant roots to soybean cyst nematode Heterodera glycines

Plant parasitic nematodes respond to root exudates to locate their host roots. In our studies second stage juveniles of Heterodera glycines, the soybean cyst nematode (SCN), quickly migrated to soybean roots in Pluronic F-127 gel. Roots of soybean and non-host Arabidopsis treated with the ethylene (ET)-synthesis inhibitor aminoethoxyvinylglycine (AVG) were more attractive to SCN than untreated roots, and significantly more nematodes penetrated into roots. Moreover, Arabidopsis ET insensitive mutants (ein2, ein2-1, ein2-5, ein3-1, ein5-1, and ein6) were more attractive than wild-type plants. Conversely, the constitutive triple-response mutant ctr1-1, was less attractive to SCN. While ET receptor gain-of-function mutant ein4-1 attracted more SCN than the wild-type, there were no significant differences in attractiveness between another gain-of-function ET receptor mutant, etr1-3, or the loss-of-function mutants etr1-7 and ers1-3 and the wild type. Expression of the reporter construct EBS: β-glucuronidase (GUS) was detected in Arabidopsis root tips as early as 6 h post infection, indicating that ET signaling was activated in Arabidopsis early by SCN infection. These results suggest that an active ET signaling pathway reduces root attractiveness to SCN in a way similar to that reported for root-knot nematodes, but opposite to that suggested for the sugar beet cyst nematode Heterodera schachtii.

Analysis of root-knot nematode and fusarium wilt disease resistance in cotton (Gossypium spp.) using chromosome substitution lines from two alien species

Chromosome substitution (CS) lines in plants are a powerful genetic resource for analyzing the contribution of chromosome segments to phenotypic variance. In this study, a series of interspecific cotton (Gossypium spp.) CS lines were used to identify a new germplasm resource, and to validate chromosomal regions and favorable alleles associated with nematode or fungal disease resistance traits. The CS lines were developed in the G. hirsutum L. TM-1 background with chromosome or chromosome segment substitutions from G. barbadense L. Pima 3–79 or G. tomentosum. Root-knot nematode (Meloidogyne incognita) and fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) (races 1 and 4) resistance alleles and quantitative trait loci (QTL) previously placed on cotton chromosomes using SSR markers in two interspecific recombinant inbred line populations were chosen for testing. Phenotypic responses of increased resistance or susceptibility in controlled inoculation and infested field assays confirmed the resistance QTLs, based on substitution with the positive or negative allele for resistance. Lines CS-B22Lo, CS-B04, and CS-B18 showed high resistance to nematode root-galling, confirming QTLs on chromosomes 4 and 22 (long arm) with resistance alleles from Pima 3–79. Line CS-B16 had less fusarium race 1-induced vascular root staining and higher percent survival than the TM-1 parent, confirming a major resistance QTL on chromosome 16. Lines CS-B(17–11) and CS-B17 had high fusarium race 4 vascular symptoms and low survival due to susceptible alleles introgressed from Pima 3–79, confirming the localization on chromosome 17 of an identified QTL with resistance alleles from TM1 and other resistant lines. Analyses validated regions on chromosomes 11, 16, and 17 harboring nematode and fusarium wilt resistance genes and demonstrated the value of CS lines as both a germplasm resource for breeding programs and as a powerful genetic analysis tool for determining QTL effects for disease resistance. CS lines carrying small alien chromosome segments with favorable QTL alleles could be used for effective introgression of biotic stress resistance or many other desirable traits by targeting gene interactions and reducing linkage drag effects.

Exogenous application of methyl jasmonate induces defence against Meloidogyne hapla in soybean

Phytohormones play important roles in plant defence against plant-parasitic nematodes, although the role of jasmonate (JA) in defence against root-knot nematodes (RKN, Meloidogyne spp.) in soybean ( Glycine max ) was unknown. In this study, two commercial soybean cultivars, cvs DongSheng1 (DS1) and SuiNong14 (SN14), were identified as susceptible and resistant, respectively, to M. hapla . Quantitative reverse transcription (qRT)-PCR analysis showed that the expression of genes involved in JA synthesis or signalling was significantly induced in both susceptible and resistant roots at 24 and 48 h after inoculation. Exogenous application of methyl jasmonate induced defence against RKN in susceptible cv. DS1, which might be involved in altered activities of defence-related enzymes (chitinase and β -1,3 glucanase) and pathogenesis-related gene PR5 expression. The results indicate that exogenous application of JA might be an alternative strategy to induce soybean resistance against RKN.

Sequence composition of BAC clones and SSR markers mapped to Upland cotton chromosomes 11 and 21 targeting resistance to soil-borne pathogens

Genetic and physical framework mapping in cotton (Gossypium spp.) were used to discover putative gene sequences involved in resistance to common soil-borne pathogens. Chromosome (Chr) 11 and its homoeologous Chr 21 of Upland cotton (G. hirsutum) are foci for discovery of resistance (R) or pathogen-induced R (PR) genes underlying QTLs involved in response to root-knot nematode (Meloidogyne incognita), reniform nematode (Rotylenchulus reniformis), Fusarium wilt (Fusarium oxysporum f.sp. vasinfectum), Verticillium wilt (Verticillium dahliae), and black root rot (Thielaviopsis basicola). Simple sequence repeat (SSR) markers and bacterial artificial chromosome (BAC) clones from a BAC library developed from the Upland cotton Acala Maxxa were mapped on Chr 11 and Chr 21. DNA sequence through Gene Ontology (GO) of 99 of 256 Chr 11 and 109 of 239 Chr 21 previously mapped SSRs revealed response elements to internal and external stimulus, stress, signaling process, and cell death. The reconciliation between genetic and physical mapping of gene annotations from new DNA sequences of 20 BAC clones revealed 467 (Chr 11) and 285 (Chr 21) G. hirsutum putative coding sequences, plus 146 (Chr 11) and 98 (Chr 21) predicted genes. GO functional profiling of Unigenes uncovered genes involved in different metabolic functions and stress response elements (SRE). Our results revealed that Chrs 11 and 21 harbor resistance gene rich genomic regions. Sequence comparisons with the ancestral diploid D5 (G. raimondii), A2 (G. arboreum) and domesticated tetraploid TM-1 AD1 (G. hirsutum) genomes revealed abundance of transposable elements and confirmed the richness of resistance gene motifs in these chromosomes. The sequence information of SSR markers and BAC clones and the genetic mapping of BAC clones provide enhanced genetic and physical frameworks of resistance gene-rich regions of the cotton genome, thereby aiding discovery of R and PR genes and breeding for resistance to cotton diseases.

QTL analysis of transgressive nematode resistance in tetraploid cotton reveals complex interactions in chromosome 11 regions

Transgressive segregation in cotton (Gossypium spp.) provides an important approach to enhance resistance to the major pest root-knot nematode (RKN) Meloidogyne incognita. Our previous studies reported transgressive RKN resistance in an intraspecific Gossypium hirsutum resistant NemX × susceptible SJ-2 recombinant inbred line (RIL) population and early generations of interspecific cross Gossypium barbadense (susceptible Pima S-7) × G. hirsutum (NemX). However, the underlying functional mechanisms for this phenomenon are not known. In this study, the region of RKN resistance gene rkn1 on chromosome (Chr) 11 and its homoeologous Chr 21 was fine mapped with G. raimondii D5 genome reference sequence. Transgressive resistance was found in the later generation of a new RIL population F2:7 (Pima S-7 × NemX) and one interspecific F2 (susceptible Pima S-7 × susceptible SJ-2). QTL analysis revealed similar contributions to root-galling and egg-production resistance phenotypes associated with SSR marker CIR316 linked to resistance gene rkn1 in NemX on Chr 11 in all seven populations analyzed. In testcross NemX × F1 (Pima S-7 × SJ-2) marker allele CIR069-271 from Pima S-7 linked to CIR316 contributed 63% of resistance to galling phenotype in the presence of rkn1. Similarly, in RIL population F2:8 (NemX × SJ-2), SJ-2 markers closely linked to CIR316 contributed up to 82% of resistance to root-galling. These results were confirmed in BC1F1 SJ-2 × F1 (NemX × SJ-2), F2 (NemX × SJ-2), and F2 (Pima S-7 × SJ-2) populations in which up to 44, 36, and 15% contribution in resistance to galling was found, respectively. Transgressive segregation for resistance was universal in all intra- and inter-specific populations, although stronger transgressive resistance occurred in later than in early generations in the intraspecific cross compared with the interspecific cross. Transgressive effects on progeny from susceptible parents are possibly provided in the rkn1 resistance region of chromosome 11 by tandemly arrayed allele (TAA) or gene (TAG) interactions contributing to transgressive resistance. Complex TAA and TAG recombination and interactions in the rkn1 resistance region provide three genes and a model to study disease and transgressive resistance in polyploid plants, and novel genotypes for plant breeding.