Pseudomonas fluorescens mediated suppression of Meloidogyne incognita infection of cowpea and tomato

Plant growth-promoting rhizobacterium, Pseudomonas fluorescens strain BICC602 suppresses root-knot nematode (Meloidogyne incognita) by enhancing defence mechanism leading to induced systemic resistance in cowpea (Vigna unguiculata) cv. L.Walp. and tomato (Solanum lycopersicum) cv. Pusa Ruby. In cowpea, the soil treatment proved more effective than foliar spray on root galling and eggs in roots. However, which factors are necessary in the induction of resistance response in plants against nematodes by BICC602 is not yet known. Salicylic acid (SA) production by some bacteria acts as endogenous signal for the activation of certain plant defence responses. In a split-root trial with tomato as a host plant and M. incognita as challenging parasite, BICC602 induces systemic resistance in tomato plants. Based on the results, it is assumed that P. fluorescens-induced resistance against M. incognita in cowpea and tomato is made either through SA-dependent or SA-independent transduction pathway.     

Isofemale Line Analysis of Meloidogyne incognita Virulence to Cowpea Resistance Gene Rk

Isofemale lines (IFL) from single egg masses were studied for genetic variation in Meloidogyne incognita isolates avirulent and virulent to the resistance gene Rk in cowpea (Vigna unguiculata). In parental isolates cultured on susceptible and resistant cowpea, the virulent isolate contained 100% and the avirulent isolate 7% virulent lineages. Virulence was selected from the avirulent isolate within eight generations on resistant cowpea (lineage selection). In addition, virulence was selected from avirulent females (individual selection). Virulence differed (P ≤ 0.05) both within and between cohorts of IFL cultured for up to 27 generations on susceptible or resistant cowpea. Distinct virulence profiles were observed among IFL. Some remained avirulent on susceptible plants and became extinct on resistant plants; some remained virulent on resistant and susceptible plants; some changed from avirulent to virulent on resistant plants; and others changed from virulent to avirulent on susceptible plants. Also, some IFL increased in virulence on susceptible plants. Single descent lines from IFL showed similar patterns of virulence for up to six generations. These results revealed considerable genetic variation in virulence in a mitotic parthenogenetic nematode population. The frequencies of lineages with stable or changeable virulence and avirulence phenotypes determined the overall virulence potential of the population.     

Fitness of Virulent Meloidogyne incognita Isolates on Susceptible and Resistant Cowpea

A study of life-history traits was made to determine factors associated with the fitness of Meloidogyne incognita isolates virulent to resistance gene Rk in cowpea. Egg hatch, root penetration, egg mass production, and fecundity (eggs per egg mass) of avirulent and virulent phenotypes were compared among M. incognita isolates, isofemale lines, and single descent lines over multiple generations on resistant and susceptible cowpea. Variation (P ≤ 0.05) in both hatch and root penetration rates was found among isolates at a given generation. However, this variation was not consistent within nematode lines among generations, and there was no correlation with level of virulence, except for penetration and virulence on resistant cowpea at generation 20. Resistant and susceptible cowpea roots were penetrated at similar levels. Differences in reproductive factors on resistant plants were correlated with levels of virulence expression. In some isofemale lines, single descent lines, and isolates, lower (P ≤ 0.05) rates of egg mass production and fecundity on susceptible cowpea were associated with virulence to Rk, indicating a trade-off between reproductive fitness and virulence. Other virulent nematode lines from the same isolates did not have reduced reproductive ability on susceptible cowpea over 27 generations. Thus, virulent lineages varied in reproductive ability on susceptible cowpea, contributing to adaptation and maintenance of virulence within M. incognita populations under stabilizing selection.     

Response of Resistant and Susceptible Bell Pepper (Capsicum annuum) to a Southern California Meloidogyne incognita Population from a Commercial Bell Pepper Field

To determine the presence and level of root-knot nematode (Meloidogyne spp.) infestation in Southern California bell pepper (Capsicum annuum) fields, soil and root samples were collected in April and May 2012 and analyzed for the presence of root-knot nematodes. The earlier samples were virtually free of root-knot nematodes, but the later samples all contained, sometimes very high numbers, of root-knot nematodes. Nematodes were all identified as M. incognita. A nematode population from one of these fields was multiplied in a greenhouse and used as inoculum for two repeated pot experiments with three susceptible and two resistant bell pepper varieties. Fruit yields of the resistant peppers were not affected by the nematodes, whereas yields of two of the three susceptible pepper cultivars decreased as a result of nematode inoculation. Nematode-induced root galling and nematode multiplication was low but different between the two resistant cultivars. Root galling and nematode reproduction was much higher on the three susceptible cultivars. One of these susceptible cultivars exhibited tolerance, as yields were not affected by the nematodes, but nematode multiplication was high. It is concluded that M. incognita is common in Southern California bell pepper production, and that resistant cultivars may provide a useful tool in a nonchemical management strategy.     

Striga gesnerioidesParasitising Cowpea: Development of Infection Structures and Mechanisms of Penetration

Striga gesnerioides(Scrophulariaceae) is an obligate hemiparasiticangiosperm, that infects the roots of cowpea (Vigna unguiculata).The development ofStrigaseedlings was characterized after transferto the surface of cowpea roots using cryo-scanning electronmicroscopy. Contact with the surface of the root caused distinctmorphological changes in theStrigaradicles. Radicle elongationceased 8 h after contact with host roots and radicle hairs developed.These hairs adhered to the surface of the host root. Early stagesof root penetration, i.e. prior to connection to the host vascularsystem were examined by light microscopy and transmission electronmicroscopy to determine the mechanisms by whichStrigainvadesroot tissue. Cowpea roots were penetrated byS. gesnerioidesradiclesafter 48 h. Host cells immediately surrounding the invadingStrigawerecompressed, but other host cells were not affected. Immunocytochemistry,using monoclonal antibodies JIM 5 and JIM 7, showed thatStrigaadvancedintercellularly through the root tissues without extensive removalof pectins at the site of penetration.Copyright 1998 Annalsof Botany Company Striga gesnerioides,Vigna unguiculata, mechanisms of penetration, host-parasite interactions, electron microscopy, immunocytochemistry.     

Characterization of Root-Knot Nematode Resistance in Medicago truncatula

Root knot (Meloidogyne spp.) and cyst (Heterodera and Globodera spp.) nematodes infect all important crop species, and the annual economic loss due to these pathogens exceeds $90 billion. We screened the worldwide accession collection with the root-knot nematodes Meloidogyne incognita, M. arenaria and M. hapla, soybean cyst nematode (SCN-Heterodera glycines), sugar beet cyst nematode (SBCN-Heterodera schachtii) and clover cyst nematode (CLCN-Heterodera trifolii), revealing resistant and susceptible accessions. In the over 100 accessions evaluated, we observed a range of responses to the root-knot nematode species, and a non-host response was observed for SCN and SBCN infection. However, variation was observed with respect to infection by CLCN. While many cultivars including Jemalong A17 were resistant to H. trifolii, cultivar Paraggio was highly susceptible. Identification of M. truncatula as a host for root-knot nematodes and H. trifolii and the differential host response to both RKN and CLCN provide the opportunity to genetically and molecularly characterize genes involved in plant-nematode interaction. Accession DZA045, obtained from an Algerian population, was resistant to all three root-knot nematode species and was used for further studies. The mechanism of resistance in DZA045 appears different from Mi-mediated root-knot nematode resistance in tomato. Temporal analysis of nematode infection showed that there is no difference in nematode penetration between the resistant and susceptible accessions, and no hypersensitive response was observed in the resistant accession even several days after infection. However, less than 5% of the nematode population completed the life cycle as females in the resistant accession. The remainder emigrated from the roots, developed as males, or died inside the roots as undeveloped larvae. Genetic analyses carried out by crossing DZA045 with a susceptible French accession, {"type":"entrez-protein","attrs":{"text":"F83005","term_id":"11352626","term_text":"pir||F83005"}}F83005, suggest that one gene controls resistance in DZA045.     

Size Differences Among Root-knot Nematodes on Resistant and Susceptible Alyceclover Genotypes

The influence of plant resistance on the size of individual root-knot nematodes was determined in greenhouse experiments. Five genotypes of alyceclover were inoculated with second-stage juveniles of Meloidogyne incognita race 3 or M. arenaria race 1. Plants were harvested at selected intervals and stained for detection of the nematodes, which were dissected from the roots. Length, width, and sagittal-sectional area of each animal were measured using an image-analysis system, and areas of nematodes in all stages were compared at different times and across alyceclover lines. Nematodes feeding on roots of resistant lines were consistently smaller than those on susceptible plants, with significant differences in growth detected after the final molt. Similar results were observed with both nematode species.     

Energetics of Meloidogyne incognita on Resistant and Susceptible Alyceclover Genotypes

To determine the energy cost of a population of Meloidogyne incognita on the roots of alyceclover, nematode biomass was estimated and equations in the literature were used to calculate energy budgets. Amounts of energy consumed, respired, or used in production of nematode biomass were calculated. Results suggested that severe infestations of root-knot nematodes can remove significant quantities of energy from their hosts. Over a 36-day period, a population of 2.6 females of M. incognita per root system removed less than 0.4 calories of energy from a resistant alyceclover plant but over 11 calories were removed by 28 females from a susceptible alyceclover. The calculations indicate that on the resistant alyceclover line, 53% of the energy assimilated by the root-knot population was allocated to respiration, with only 47% allocated to production, whereas on the susceptible line, 65% of the assimilated energy was allocated to production. Such energy demands by the parasite could result in significant reductions in yield quantity and quality at a field production level.     

<Emphasis Type="Italic">CaMi</Emphasis>, a root-knot nematode resistance gene from hot pepper (<Emphasis Type="Italic">Capsium annuum</Emphasis> L.) confers nematode resistance in tomato

Several root-knot nematode (Meloidogyne spp.) resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, none of them has yet been cloned. In this study, a candidate root-knot nematode resistance gene (designated as CaMi) was isolated from the resistant pepper line PR 205 by degenerate PCR amplification combined with the RACE technique. Expression profiling analysis revealed that this gene was highly expressed in roots, leaves, and flowers and expressed at a lower level in stems and was not detectable in fruits. To verify the function of CaMi, a sense vector containing the genomic DNA spanning the full coding region of CaMi was constructed and transferred into root-knot nematode susceptible tomato plants. Sixteen transgenic plants carrying one to five copies of T-DNA inserts were generated from two nematode susceptible tomato cultivars. RT-PCR analysis revealed that the expression levels of CaMi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some transgenic lines compared to untransformed susceptible plants, and that the resistance was inheritable. Ultrastructure analysis showed that nematodes led to the formation of galls or root knots in the susceptible lines while in the resistant transgenic plants, the CaMi gene triggered a hypersensitive response (HR) as well as many necrotic cells around nematodes. Rugang Chen and Hanxia Li are contributed equally to this work.     

Global changes in gene expression during compatible and incompatible interactions of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides

ABSTRACT: BACKGROUND: Cowpea, Vigna unguiculata L. Walp., is one of the most important food and forage legumesin the semi-arid tropics. While most domesticated forms of cowpea are susceptible to the rootparasitic weed Striga gesnerioides, several cultivars have been identified that show racespecificresistance. Cowpea cultivar B301 contains the RSG3-301 gene for resistance to S.gesnerioides race SG3, but is susceptible to race SG4z. When challenged by SG3, roots ofcultivar B301 develop a strong resistance response characterized by a hypersensitive reactionand cell death at the site of parasite attachment. In contrast, no visible response occurs inB301 roots parasitized by SG4z. RESULTS: Gene expression in the roots of the cowpea cultivar B301 during compatible (susceptible) andincompatible (resistant) interactions with S. gesnerioides races SG4z and SG3, respectively,were investigated at the early (6 days post-inoculation (dpi)) and late (13 dpi) stages of theresistance response using a Nimblegen custom design cowpea microarray. A total of 111genes were differentially expressed in B301 roots at 6 dpi; this number increased to 2102genes at 13 dpi. At 13 dpi, a total of 1944 genes were differentially expressed duringcompatible (susceptible) interactions of B301 with SG4z . Genes and pathways involved insignal transduction, programmed cell death and apoptosis, and defense response to biotic andbiotic stress were differentially expressed in the early resistance response; at the later timepoint, enrichment was primarily for defense-related gene expression, and genes encodingcomponents of lignifications and secondary wall formation. In compatible interactions (B301- SG4z), multiple defense pathways were repressed, including those involved in ligninbiosynthesis and secondary cell wall modifications, while cellular transport processes fornitrogen and sulfur were increased. CONCLUSION: Distinct changes in global gene expression profiles occur in host roots following successfuland unsuccessful attempted parasitism by Striga. Induction of specific defense related genesand pathways defines components of a unique resistance mechanism. Some genes andpathways up-regulated in the host resistance response to SG3 are repressed in the susceptibleinteractions, suggesting that the parasite is targeting specific components of the host'sdefense. These results add to our understanding of plant-parasite interactions and theevolution of resistance to parasitic weeds.     

Differential expression of root-knot nematode resistance genes in tomato and pepper: evidence with Meloidogyne incognita virulent and avirulent near-isogenic lineages

Reproduction of artificially selected near isogenic Meloidogyne incognita lineages virulent and avirulent against the Mi resistance gene of tomato was assessed on host and resistant lines and cultivars of pepper. Egg mass production following inoculation of individual potted seedlings with second-stage juveniles was studied in experiments conducted in controlled environment. Artificially selected Mi-virulent nematode populations were unable to develop on resistant pepper lines PM 217 and PM 687. This suggests that the genetic systems governing resistance to root-knot nematodes are differently expressed in tomato and pepper, in spite of the very close phylogenetic relationships and structural genomic homologies occurring between these two vegetable crops. Moreover, these artificially selected nematode populations were also found unable to develop on the susceptible pepper cultivars California Wonder and Doux Long des Landes, while their pathogenicity was not significantly affected on susceptible tomatoes. Due to the existence of naturally virulent Meloidogyne populations, these results enhance the need for a better understanding of the mechanisms involved, in order to develop new forms of management of plant resistance to root-knot nematodes.     

Determination of Lignin and Tannin Contents of Cowpea Seed Coats

Tannins in the seed coats of cowpea cultivars interfere withthe determination of lignin by the acetyl bromide method butthe tannins can be removed by extraction with acetone:water(70:30) allowing a direct determination on the residue. Furthermore,the difference between the absorbance values determined by theacetyl bromide method before and after the removal of tanninscan be used as a measure of tannin content in cowpea seed coats.Copyright1995, 1999 Academic Press Cowpea, Vigna unguiculata, lignin, tannin, acetyl bromide     

Influence of Soil Temperature on Meloidogyne incognita Resistant and Susceptible Cotton,Gossypium hirsutum

The degree of resistance by a cotton plant to Meloidogyne incognita is affected by soil temperature, particularly in moderately resistant cultivars, The total number of nematodes in the resistant and moderately resistant rools at 35 C was equal to, or greater than, the number in susceptible roots at 20, 25, or 30 C. A shift in numbers to developing and egg-bearing forms of nematodes in the susceptible cultivar as tentperature increased indicates development was affected by temperature rather than by genetic resistance mechanisms. However, the nematode resistant cultivar did not support maturation of nematodes until a soil tempurature of 35 C was attained. This indicated that resistance mechanisms are partially repressed at 35 C and differences in nematode development cannot be explained in terms of accumulated heat units. The moderately resistant cultivar was significantly more sensitive to the effects of high temperature than was the resistant cultivar.     

Interaction of Vesicular-Arbuscular Mycorrhizae and Cultivars of Alfalfa Susceptible and Resistant to Meloidogyne hapla

The interaction between vesicular-arbuscular mycorrhizal (VAM) fungi and the root-knot nematode (Meloidogyne hapla) was investigated using both nematode-susceptible (Grasslands Wairau) and nematode-resistant (Nevada Synthetic XX) cultivars of alfalfa (Medicago sativa) at four levels of applied phosphate. Mycorrhizal inoculation improved plant growth and reduced nematode numbers and adult development in roots in dually infected cultures of the susceptible cultivar. The tolerance of plants to nematode infection and development when preinfected with mycorrhizal fungi was no greater than when they were inoculated with nematodes and mycorrhizal fungi simultaneously. Growth of plants of the resistant cultivar was unaffected by nematode inoculation but was improved by mycorrhizal inoculation. Numbers of nematode juveniles were lower in the roots of the resistant than of the susceptible cultivar and were further reduced by mycorrhizal inoculation, although no adult nematodes developed in any resistant cultivar treatment. Inoculation of alfalfa with VAM fungi increased the tolerance and resistance of a cultivar susceptible to M. hapla and improved the resistance of a resistant cultivar.     

Host Plant Resistance as an Alternative to Methyl Bromide for Managing Meloidogyne incognita in Pepper

Pre-plant soil fumigation with methyl bromide and host resistance were compared for managing the southern root-knot nematode (Meloidogyne incognita) in pepper. Three pepper cultivars (Carolina Cayenne, Keystone Resistant Giant, and California Wonder) that differed in resistance to M. incognita were grown in field plots that had been fumigated with methyl bromide (98% CH₃Br : 2% CCl₃NO₂ [w/w]) before planting or left untreated. Carolina Cayenne is a well-adapted cayenne-type pepper that is highly resistant to M. incognita. The bell-type peppers Keystone Resistant Giant and California Wonder are intermediate to susceptible and susceptible, respectively. None of the cultivars exhibited root galling in the methyl bromide fumigated plots and nematode reproduction was minimal (<250 eggs/g fresh root), indicating that the fumigation treatment was highly effective in controlling M. incognita. Root galling of Carolina Cayenne and nematode reproduction were minimal, and fruit yields were not reduced in the untreated plots. The root-galling reaction for Keystone Resistant Giant was intermediate (gall index = 2.9, on a scale of 1 to 5), and nematode reproduction was moderately high. However, yields of Keystone Resistant Giant were not reduced in untreated plots. Root galling was severe (gall index = 4.3) on susceptible California Wonder, nematode reproduction was high, and fruit yields were reduced (P ≤ 0.05) in untreated plots. The resistance exhibited by Carolina Cayenne and Keystone Resistant Giant provides an alternative to methyl bromide for reducing yield losses by southern root-knot nematodes in pepper. The high level of resistance of Carolina Cayenne also suppresses population densities of M. incognita.     

Pepper Rootstock Graft Compatibility and Response to Meloidogyne javanica and M. incognita

Resistance of pepper species (Capsicum annuum, C. baccatum, C. chinense, C. chacoense, and C. frutescens), cultivars and accessions to the root-knot nematodes Meloidogyne incognita race 2 and M. javanica, and their graft compatibility with commercial pepper varieties as rootstocks were evaluated in growth chamber and greenhouse experiments. Most of the plants tested were highly resistant to M. javanica but susceptible to M. incognita. Capsicum annuum AR-96023 and C. frutescens accessions as rootstocks showed moderate and relatively high resistance to M. incognita, respectively. In M. incognita-infested soil in a greenhouse, AR-96023 supported approximately 6-fold less nematode eggs per gram root and produced about 2-fold greater yield compared to a nongrafted commercial variety. The commercial variety grafted on AR-96023 produced a yield as great as the non-grafted variety in the root-knot nematode-free greenhouse. Some resistant varieties and accessions used as rootstocks produced lower yields (P < 0.01) than that of the non-grafted variety in the noninfested greenhouse. Use of rootstocks with nematode-resistance and graft compatibility may be effective for control of root-knot nematodes on susceptible pepper.     

Potential of Tissue Culture for Breeding Root-Knot Nematode Resistance into Vegetables

Plant protoplast technology is being investigated as a means of transferring root-knot nematode resistance factors from Solanum sisymbriifolium into the susceptible S. melongena. Solanum sisymbriifolium plants regenerated from callus lost resistance to Meloidogyne javanica but retained resistance to M. incognita. Tomato plants cloned from leaf discs of the root-knot nematode resistant ''Patriot'' were completely susceptible to M. incognita, while sections of stems and leaves rooted in sand in the absence of growth hormones retained resistance. Changes in resistance persisted for three generations. It is postulated that the exogenous hormonal constituents of the culture medium are modifying the expression of genetic resistance.     

Effects of Tomato Root Exudates on Meloidogyne incognita

Plant root exudates affect root-knot nematodes egg hatch. Chemicals in root exudates can attract nematodes to the roots or result in repellence, motility inhibition or even death. However, until recently little was known about the relationship between tomato root exudates chemicals and root-knot nematodes. In this study, root exudates were extracted from three tomato rootstocks with varying levels of nematode resistance: Baliya (highly resistant, HR), RS2 (moderately resistant, MR) and L-402 (highly susceptible, T). The effects of the root exudates on Meloidogyne incognita (M. incognita) egg hatch, survival and chemotaxis of second-stage juveniles (J2) were explored. The composition of the root exudates was analysed by gas chromatography/mass spectrometry (GC/MS) prior to and following M. incognita inoculation. Four compounds in root exudates were selected for further analysis and their allopathic effect on M. incognita were investigated. Root exudates from each tomato rootstocks (HR, MR and T strains) suppressed M. incognita egg hatch and increased J2 mortality, with the highest rate being observed in the exudates from the HR plants. Exudate from HR variety also repelled M. incognita J2 while that of the susceptible plant, T, was demonstrated to be attractive. The relative amount of esters and phenol compounds in root exudates from HR and MR tomato rootstocks increased notably after inoculation. Four compounds, 2,6-Di-tert-butyl-p-cresol, L-ascorbyl 2,6-dipalmitate, dibutyl phthalate and dimethyl phthalate increased significantly after inoculation. The egg hatch of M. incognita was suppressed by each of the compound. L-ascorbyl 2,6-dipalmitate showed the most notable effect in a concentration-dependent manner. All four compounds were associated with increased J2 mortality. The greatest effect was observed with dimethyl phthalate at 2 mmol·L^-1. Dibutyl phthalate was the only compound observed to repel M. incognita J2 with no effect being detected in the other compounds. Each of the four compounds were correlated with a reduction in disease index in the susceptible cultivar, T, and tomato seedlings irrigated with L-ascorbyl 2,6-dipalmitate at 2 mmol·L^-1 showed the best resistance to M. incognita. Taken together, this study provided a valuable contribution to understanding the underlying mechanism of nematode resistance in tomato cultivars.