Molecular Transfer of Nematode Resistance Genes

Recombinant DNA techniques have been used to introduce agronomically valuable traits, including resistance to viruses, herbicides, and insects, into crop plants. Introduction of these genes into plants frequently involves Agrobacterium-mediated gene transfer. The potential exists for applying this technology to nematode control by introducing genes conferring resistance to nematodes. Transferred genes could include those encoding products detrimental to nematode development or reproduction as well as cloned host resistance genes. Host genes that confer resistance to cyst or root-knot nematode species have been identified in many plants. The best characterized is Mi, a gene that confers resistance to root-knot nematodes in tomato. A map-based cloning approach is being used to isolate the gene. For development of a detailed map of the region of the genome surrounding Mi, DNA markers genetically linked to Mi have been identified and analyzed in tomato lines that have undergone a recombination event near Mi. The molecular map will be used to identify DNA corresponding to Mi. We estimate that a clone of Mi will be obtained in 2-5 years. An exciting prospect is that introduction of this gene will confer resistance in plant species without currently available sources of resistance.     

Reproduction of Mi-Virulent Meloidogyne incognita Isolates on Lycopersicon spp.

Selection of detectable numbers of Mi-virulent root-knot nematodes has necessitated a greater understanding of nematode responses to new sources of resistance. During the course of this research, we compared the reproduction of four geographically distinct Mi-virulent root-knot nematode isolates on three resistant accessions of Lycopersicon peruvianum. Each accession carried a different resistant gene, Mi-3, Mi-7, or Mi-8. All nematode isolates were verified as Meloidogyne incognita using diagnostic markers in the mitochondrial genome of the nematode. Reproduction of Mi-virulent isolates W1, 133 and HM, measured as eggs per g of root, was greatest on the Mi-7 carrying accession and least on the Mi-8 carrying accession. In general, Mi-3 behaved similar to the Mi-8 carrying accession. Reproduction of the four nematode isolates was also compared on both Mi and non-Mi-carrying L. esculentum cultivars and a susceptible L. peruvianum accession. Resistance mediated by Mi in L. esculentum still impacted the Mi-virulent nematodes with fewer eggs per g of root on the resistant cultivar (P ≤ 0.05). Preliminary histological studies suggests that Mi-8 resistance is mediated by a hypersensitive response, similar to Mi.     

Evidence for a Dosage Effect of the Mi gene on Partially Virulent Isolates of Meloidogyne javanica

The reproduction of single egg-mass isolates of Meloidogyne javanica from Crete that differed in virulence were compared on tomato (Lycopersicon esculentum) genotypes homozygous or heterozygous for the Mi gene. The reproduction of three isolates with partial virulence was much greater on tomato genotypes heterozygous for the Mi gene (cultivars Scala, Bermuda, and 7353) than on two homozygous genotypes (F8 inbred lines derived from Scala). The reproduction of a highly virulent isolate on the homozygous and heterozygous genotypes was similar to that on a susceptible cultivar. These results pose questions regarding the nature of partial virulence and indicate a quantitative effect of the Mi gene in relation to such virulence.     

Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant

RNA interference (RNAi)-based host-induced gene silencing (HIGS) is emerging as a novel, efficient and target-specific tool to combat phytonematode infection in crop plants. Mi-msp-1, an effector gene expressed in the subventral pharyngeal gland cells of Meloidogyne incognita plays an important role in the parasitic process. Mi-msp-1 effector is conserved in few of the species of root-knot nematodes (RKNs) and does not share considerable homology with the other phytonematodes, thereby making it a suitable target for HIGS with minimal off-target effects. Six putative eggplant transformants harbouring a single copy RNAi transgene of Mi-msp-1 was generated. Stable expression of the transgene was detected in T₁, T₂ and T₃ transgenic lines for which a detrimental effect on RKN penetration, development and reproduction was documented upon challenge infection with nematode juveniles. The post-parasitic nematode stages extracted from the transgenic plants showed long-term RNAi effect in terms of targeted downregulation of Mi-msp-1. These findings suggest that HIGS of Mi-msp-1 enhances nematode resistance in eggplant and protect the plant against RKN parasitism at very early stage.

     

The effector protein Avr2 of the xylem-colonizing fungus Fusarium oxysporum activates the tomato resistance protein I-2 intracellularly

To promote host colonization, many plant pathogens secrete effector proteins that either suppress or counteract host defences. However, when these effectors are recognized by the host's innate immune system, they trigger resistance rather than promoting virulence. Effectors are therefore key molecules in determining disease susceptibility or resistance. We show here that Avr2, secreted by the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici ( Fol ), shows both activities: it is required for full virulence in a susceptible host and also triggers resistance in tomato plants carrying the resistance gene I-2 . Point mutations in AVR2 , causing single amino acid changes, are associated with I-2 -breaking Fol strains. These point mutations prevent recognition by I-2 , both in tomato and when both genes are co-expressed in leaves of Nicotiana benthamiana . Fol strains carrying the Avr2 variants are equally virulent, showing that virulence and avirulence functions can be uncoupled. Although Avr2 is secreted into the xylem sap when Fol colonizes tomato, the Avr2 protein can be recognized intracellularly by I-2, implying uptake by host cells.     

Effect of the Mi Gene in Tomato on Reproductive Factors of Meloidogyne chitwoodi and M. hapla

The effect of the Mi gene on the reproductive factor of Meloidogyne chitwoodi and M. hapla, major nematode pests of potato, was measured on nearly isogenic tomato lines differing in presence or absence of the Mi gene. The Mi allele controlled resistance to reproduction of race 1 of M. chitwoodi and to one of two isolates of race 2. No resistance to race 3 of M. chitwoodi or to M. hapla was found. Variability in response to isolates of race 2 may reflect diversity of virulence genotypes heretofore undetected. Resistance to race 1 of M. chitwoodi could be useful in potato if the Mi gene were functional following transferral by gene insertion technology into potato. Since the Mi gene is not superior to RMc₁ derived from Solarium bulbocastanum, the transferral by protoplast fusion appears to offer no advantage.     

Genetic and physical localization of the root-knot nematode resistance locus Mi in tomato

As part of a map-based cloning strategy designed to isolate the root-knot nematode resistance gene Mi, tomato F2 populations were analyzed in order to identify recombination points close to this economically important gene. A total of 21?089 F2 progeny plants were screened using morphological markers. An additional 1887 F2 were screened using PCR-based flanking markers. Fine-structure mapping of recombinants with newly developed AFLP markers, and RFLP markers derived from physically mapped cosmid subclones, localized Mi to a genomic region of about 550?kb. The low frequency of recombinants indicated that recombination was generally suppressed in these crosses and that crossovers were restricted to particular regions. To circumvent this problem, a population of Lycopersicon peruvianum, the species from which Mi was originally introgressed, that was segregating for resistance was developed. Screening of this population with PCR, RFLP and AFLP markers identified several plants with crossovers near Mi. Recombination frequency was approximately eight-fold higher in the Mi region of the L. peruvianum cross. However, even within the wild species cross, recombination sites were not uniformly distributed in the region. By combining data from the L.?esculentum and L. peruvianum recombinant analyses, it was possible to localize Mi to a region of the genome spanning less than 65?kb.     

Isolation and characterization of a tomato Acid phosphatase complementary DNA associated with nematode resistance

The tomato (Lycopersicon esculentum) acid phosphatase-1 (Apase-1¹, EC 3.1.3.2) isozyme variant, genetically linked to the root-knot nematode resistance locus (Mi) on chromosome 6, has been purified by a rapid procedure from tomato cell suspension cultures. Peptide fragments of the purified enzyme were generated from trypsin and Lys-C endoprotease digests and separated by reverse-phase high-performance liquid chromatography. Amino acid sequences derived from the purified peptide fragments represented >50% of the total amino acid content of the protein and enabled the construction of degenerate oligonucleotide probes that were used to screen a tomato cell culture complementary DNA library. Clones corresponding to full-length coding sequences for Apase-1 have been isolated and sequenced. Southern blot analysis of DNA isolated from a number of tomato cultivars shows that the Apase-1¹ gene (aps1) is present at one copy per genome and that genotypes containing the aps1¹ allele have restriction fragment length polymorphisms that distinguish them from cultivars having the aps1⁺ allele. Segregation analysis demonstrates that the restriction fragment length polymorphisms are associated with the aps1 locus. Tomato Apase-1¹ is also found to have significant homology at the amino acid sequence level to a class of vegetative storage proteins characterized in soybean.     

Reproduction of Virulent Isolates of Meloidogyne incognita on Susceptible and Mi-resistant Tomato

The reproductive potential of natural and laboratory-selected Meloidogyne incognita isolates virulent against the tomato Mi resistance gene, all derived from a single egg-mass, were compared when the nematodes were inoculated on susceptible and resistant tomato. Fewer second-stage juveniles (P = 0.01) of the two virulent populations selected under laboratory conditions matured to females on the resistant tomato compared to the susceptible cultivar. In contrast, no differences were found between the number of egg masses produced on the resistant versus the susceptible tomato by the two natural virulent isolates. No clear general trends concerning the fecundity of the females could be inferred from the comparative analysis of the numbers of eggs per egg mass x tomato cultivar combination. These observations suggested that the genetic changes induced under environmentally controlled nematode growth might be different from those occurring in natural Mi-resistance breaking biotypes grown without environmental control.     

Mapping a new nematode resistance locus in Lycopersicon peruvianum

Accessions of the wild tomato species L. peruvianum were screened with a root-knot nematode population (557R) which infects tomato plants carrying the nematode resistance gene Mi. Several accessions were found to carry resistance to 557R. A L. peruvianum backcross population segregating for resistance to 557R was produced. The segregation ratio of resistant to susceptible plants suggested that a single, dominant gene was a major factor in the new resistance. This gene, which we have designated Mi-3, confers resistance against nematode strains that can infect plants carrying Mi. Mi-3, or a closely linked gene, also confers resistance to nematodes at 32°C, a temperature at which Mi is not effective. Bulked-segregant analysis with resistant and susceptible DNA pools was employed to identify RAPD markers linked to this gene. Five-hundred-and-twenty oligonucleotide primers were screened and two markers linked to the new resistance gene were identified. One of the linked markers (NR14) was mapped to chromosome 12 of tomato in an L. esculentum/L. pennellii mapping population. Linkage of NR14 and Mi-3 with RFLP markers known to map on the short arm of chromosome 12 was confirmed by Southern analysis in the population segregating for Mi-3. We have positioned Mi-3 near RFLP marker TG180 which maps to the telomeric region of the short arm of chromosome 12 in tomato.     

A PCR-based marker tightly linked to the nematode resistance gene,Mi, in tomato

A PCR-based codominant marker has been developed which is tightly linked to Mi, a dominant genetic locus in tomato that confers resistance to several species of root-knot nematode. DNA from tomato lines differing in nematode resistance was screened for random amplified polymorphic DNA markers linked to Mi using decamer primers. Several markers were identified. One amplified product, REX-1, obtained using a pair of decamer primers, was present as a dominant marker in all nematode-resistant tomato lines tested. REX-1 was cloned and the DNA sequences of its ends were determined and used to develop 20-mer primers. PCR amplification with the 20-mer primers produced a single amplified band in both susceptible and resistant tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq I. The linkage of REX-1 to Mi was verified in an F₂ population. This marker is more tightly linked to Mi than is Aps-1, the currently-used isozyme marker, and allows screening of germplasm where the linkage between Mi and Aps-1 has been lost. Homozygous and heterozygous individuals can be distinguished and the procedure can be used for rapid, routine screening. The strategy used to obtain REX-1 is applicable to obtaining tightly-linked markers to other genetic loci. Such markers would allow rapid, concurrent screening for the segregation of several loci of interest.     

Effects of the Mi-1, N and Tabasco Genes on Infection and Reproduction of Meloidogyne mayaguensis on Tomato and Pepper Genotypes

Meloidogyne mayaguensis is a damaging root-knot nematode able to reproduce on root-knot nematode-resistant tomato and other economically important crops. In a growth chamber experiment conducted at 22 and 33°C, isolate 1 of M. mayaguensis reproduced at both temperatures on the Mi-1-carrying tomato lines BHN 543 and BHN 585, whereas M. incognita race 4 failed to reproduce at 22°C, but reproduced well at 33°C. These results were confirmed in another experiment at 26 ± 1.8°C, where minimal or no reproduction of M. incognita race 4 was observed on the Mi-1-carrying tomato genotypes BHN 543, BHN 585, BHN 586 and ‘Sanibel’, whereas heavy infection and reproduction of M. mayaguensis isolate 1 occurred on these four genotypes. Seven additional Florida M. mayaguensis isolates also reproduced on resistant ‘Sanibel’ tomato at 26 ± 1.8°C. Isolate 3 was the most virulent, with reproduction factor (Rf) equal to 8.4, and isolate 8 was the least virulent (Rf = 2.1). At 24°C, isolate 1 of M. mayaguensis also reproduced well (Rf ≥ 1) and induced numerous small galls and large egg masses on the roots of root-knot nematode-resistant bell pepper ‘Charleston Belle’ carrying the N gene and on three root-knot nematode-resistant sweet pepper lines (9913/2, SAIS 97.9001 and SAIS 97.9008) carrying the Tabasco gene. In contrast, M. incognita race 4 failed to reproduce or reproduced poorly on these resistant pepper genotypes. The ability of M. mayaguensis isolates to overcome the resistance of tomato and pepper genotypes carrying the Mi-1, N and Tabasco genes limits the use of resistant cultivars to manage this nematode species in infested tomato and pepper fields in Florida.     

Management of Fusarium oxysporum f. sp. lycopersici and root-knot nematode disease complex in tomato by use of antagonistic fungi,plant resistance and neem

Simultaneous infestation with root-knot nematodes (RKN) and Fusarium oxysporum f. sp. lycopersici (FOL) leads to formation of a disease complex that increases crop losses than effect of either RKN or FOL. In this study a management programme involving plant resistance, biological control agents, and neem was carried out to manage RKN and fusarium wilt disease complex. The biological control agents were Purpureocillium lilacinum (PL) and Trichoderma harzianum (TH) while the RKN was Meloidogyne javanica. In vitro dual culture plates were set up to test the interaction of biological control agents and FOL. Greenhouse experiments were conducted using two tomato cultivars Rambo F1 and Prostar F1. The treatments were; PL, TH, PL–TH, neem, PL neem, TH neem, and PL–TH neem. Each treatment was replicated four times and the treatments set up in a randomised complete block design in the greenhouse. Inhibition of FOL mycelial growth by TH and PL was 51.9%, and 44% respectively by the ninth day in vitro culture plates. In the cultivar, Prostar F1, the treatments PL–TH, PL, and TH in the presence or absence of neem had a FOL disease severity score significantly lower than the untreated control. Host resistance sufficed to prevent infection of Rambo F1 with FOL. The treatments PL–TH, PL and TH reduced FOL propagules and M. javanica juveniles in the roots and performed even better when combined with neem in both tomato cultivars. Therefore, a host that is resistant combined with biological control agents and organic amendments can be used in the management of RKN and FOL in tomato production.     

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.     

Pasteuria penetrans for Control of Meloidogyne incognita on Tomato and Cucumber,and M. arenaria on Snapdragon

Meloidogyne incognita and Meloidogyne arenaria are important parasitic nematodes of vegetable and ornamental crops. Microplot and greenhouse experiments were conducted to test commercial formulations of the biocontrol agent Pasteuria penetrans for control of M. incognita on tomato and cucumber and M. arenaria on snapdragon. Three methods of application for P. penetrans were assessed including seed, transplant, and post-plant treatments. Efficacy in controlling galling and reproduction of the two root-knot nematode species was evaluated. Seed treatment application was assessed only for M. incognita on cucumber. Pasteuria treatment rates of a granular transplant formulation ranged from 1.5 × 10⁵ endospores/cm³ to 3 × 10⁵ endospores/cm³ of transplant mix applied at seeding. Additional applications of 1.5 × 10⁵ endospores/cm³ of soil were applied as a liquid formulation to soil post-transplant for both greenhouse and microplot trials. In greenhouse cucumber trials, all Pasteuria treatments were equivalent to steamed soil for reducing M. incognita populations in roots and soil, and reducing nematode reproduction and galling. In cucumber microplot trials there were no differences among treatments for M. incognita populations in roots or soil, eggs/g root, or root condition ratings. Nematode reproduction on cucumber was low with Telone II and with the seed treatment plus post-plant application of Pasteuria, which had the lowest nematode reproduction. However, galling for all Pasteuria treatments was higher than galling with Telone II. Root-knot nematode control with Pasteuria in greenhouse and microplot trials varied on tomato and snapdragon. Positive results were achieved for control of M. incognita with the seed treatment application on cucumber.     

南方根结线虫对不同砧木嫁接番茄苗活性氧清除系统的影响

采用盆栽人工接种方法,对番茄嫁接苗进行了抗性评价,研究了番茄嫁接苗叶片中抗氧化酶活性和活性氧代谢的动态变化。结果表明,接种南方根结线虫(J2)后,砧木嫁接苗表现为高抗,自根嫁接苗为高感。通过嫁接换根,与自根嫁接苗相比,砧木嫁接苗明显提高了接穗叶片的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性,降低了超氧阴离子(O.2-)产生速率以及过氧化氢(H2O2)和丙二醛(MDA)含量。表明番茄植株体内的活性氧水平和抗氧化酶活性的高低与其抗根结线虫的能力密切相关,较低的活性氧水平和较高的抗氧化酶活性有利于减轻对膜系统的伤害,提高番茄植株的抗根结线虫能力。     

Pathogenicity of Pochonia species on eggs of Meloidogyne javanica

Among fungi, species of the genus Pochonia Batista & O.M. Fonseca are considered as promising biological control agents with high potential to reduce root-knot nematode (RKN) and nematode populations. In this research we investigated Fars province of Iran for the presence of Pochonia spp., compared pathogenicity of different Pochonia species on eggs of RKN in vitro, and selected the best isolates for further studies. During 2004-2006, 128 soil samples of fields infested with cyst nematodes and 18 soil samples infested with RKN were collected from Fars province of Iran. In vitro pathogenicity tests were carried out on 36 isolates of Pochonia spp. obtained from CBS and IRAN culture collections. The seven best isolates of this experiment were selected for greenhouse test and their ability in controlling RKN was examined in natural soil. In greenhouse test fresh weight of plant’s tops and roots, gall index, nematode multiplication, second-stage juveniles’ population in soil, reproduction rate (P_f/P_i), proportion of infected eggs, control efficacy, root colonization and soil colony forming units were determined. In vitro pathogenicity of Pochonia on RKN eggs varied between 39% and 95% eggs infected. In greenhouse experiment, three isolates are promising for control of RKN and selected isolates are subjected to more extensive testing to determine their effectiveness in a range of conditions before being developed as commercial biological control agents.     

Reducing Meloidogyne incognita Injury to Cucumber in a Tomato-Cucumber Double-Cropping System

The effects of a root-knot nematode-resistant tomato cultivar and application of the nematicide ethoprop on root-knot nematode injury to cucumber were compared in a tomato-cucumber double-cropping system. A root-knot nematode-resistant tomato cultivar, Celebrity, and a susceptible cultivar, Heatwave, were grown in rotation with cucumber in 1995 and 1996. Celebrity suppressed populations of Meloidogyne incognita in the soil and resulted in a low root-gall rating on the subsequent cucumber crop. Nematode population densities were significantly lower at the termination of the cucumber crop in plots following Celebrity than in plots following Heatwave. Premium and marketable yields of cucumbers were higher in plots following Celebrity than in plots following Heatwave. Application of ethoprop through drip irrigation at 4.6 kg a.i./ha reduced root galling on the cucumber crop but had no effect on the nematode population density in the soil at crop termination. Ethoprop did not affect cucumber yield. These results indicate that planting a resistant tomato cultivar in a tomato-cucumber double-cropping system is more effective than applying ethoprop for managing M. incognita.     

A differential interaction study of Bemisia tabaci Q-biotype on commercial tomato varieties with or without the Mi resistance gene, and comparative host responses with the B-biotype

Three tomato varieties (Motelle, Ronita, and VFN8) bearing the Mi-1.2 gene providing resistance to nematodes Meloidogyne spp. and to the potato aphid Macrosiphum euphorbiae Thomas, and three varieties not bearing this gene (Moneymaker, Roma, and Río Fuego), were compared by choice assay for host preference using the Q-biotype of Bemisia tabaci (Gennadius). The most preferred hosts, determined by infestation levels and numbers of feeding adults were Moneymaker, Río Fuego and Roma, all of which were not carrying the Mi gene. Ronita and Motelle, both of which bore the Mi gene, were the least preferred hosts. In a no-choice assay, B. tabaci females laid a significantly lower number of eggs on the varieties that carried the Mi gene than on those lacking the gene. Differences were more dramatic when plants carrying the Mi gene were pooled together and compared with pooled plants without this gene. Significantly greater values were obtained for the Mi-lacking group for all parameters tested. Comparing these results with those from a previous study on the B-biotype of B. tabaci, Q-biotypes were found to produce higher daily infestation rates on most of the tomato varieties. When results from plants carrying Mi were pooled, they showed lower infestation levels of Q-biotypes than B-biotypes. The Q-biotype infested less Mi-plants and more non-Mi plants than B-biotype. Q-biotype females produced significantly less pupae than the B-biotype females on both groups of plants. These results suggest the existence of an antixenosis and antibiosis-based resistance to the Q-biotype of B. tabaci in Mi-bearing commercial tomato varieties, which is greater than that previously reported for the B-biotype.