Correlation of genetic and physical structure in the region surrounding the I2Fusarium oxysporum resistance locus in tomato

Summary The dominant gene I ₂ confers on tomato (Lycopersicon esculentum) resistance against the fungus Fusarium oxysporum f. sp. lycopersici race 2. A restriction fragment length polymorphism (RFLP) marker, TG105, has recently been found to be tightly linked to I ₂. The potential for cloning this gene by a reverse genetics approach prompted us to describe in both genetic and physical detail the region surrounding the I ₂ locus on chromosome 11. We have analyzed patterns of segregation of RFLP markers on chromosome 11 and Fusarium resistance in 140 F₂ plants from a cross between Fusarium-resistant and susceptible parental lines. Marker TG105 mapped 0.4 centi-Morgan (CM) from I ₂. Physical analysis of TG105 and its flanking RFLP markers, TG26 and TG36, by pulsed field gradient gel electrophoresis (PFGE) yielded a restriction map for this region encompassing at least 620 kb of the tomato genome. TG105 and TG26 hybridized to the same 175 kb MluI-NruI restriction fragment. We have therefore linked two genetically distinct RFLP markers. Based on the 4.1 cM distance between them, we have assigned a mean value of 43 kb for each cM recombination distance in the vicinity of I ₂. This local ratio between physical and genetic distances is more than 10-fold below the average for the tomato genome. It should therefore be possible to clone I ₂ by chromosome walking from TG105.     

An RFLP marker in tomato linked to the Fusarium oxysporum resistance gene I2

Summary The locus, I2, which in tomato confers resistance against Fusarium oxysporum f. sp. lycopersici race 2, was introgressed into Lycopersicon esculentum from the wild species L. pimpinellifolium (P.I. 126915). We searched for restriction fragment length polymorphisms (RFLPs) between nearly isogenic lines (NILs) in clones that map to the region introgressed from the wild species. Since I2 maps to chromosome 11, we used DNA clones from this chromosome as hybridization probes to Southern blots containing bound DNA of the NILs digested with 23 restriction enzymes. Of the 14 chromosome 11 clones, 9 exhibited polymorphism. These clones were further hybridized to verification filters that contained DNA from resistant and susceptible L. esculentum varieties digested with the enzymes that gave the polymorphism. One clone, TG105, was found to be associated with I2; 19 susceptible lines showed a different RFLP with this probe than 16 resistant lines, including the original L. pimpinellifolium accession used as a source for the resistance gene. These results together with our mapping analysis indicate that TG105 is closely linked to the resistance gene.     

RFLP mapping of I1, a new locus in tomato conferring resistance against Fusarium oxysporum f. sp. lycopersici race 1

Summary The inheritance and linkage relationships of a gene for resistance to Fusarium oxysporum f. sp. lycopersici race 1 were analyzed. An interspecific hybrid between a resistant Lycopersicon pennellii and a susceptible L. esculentum was backcrossed to L. esculentum. The genotype of each backcross-1 (BC₁) plant with respect to its Fusarium response was determined by means of backcross-2 progeny tests. Resistance was controlled by a single dominant gene, I1, which was not allelic to I, the traditional gene for resistance against the same fungal pathogen that was derived from L. pimpinellifolium. Linkage analysis of 154 molecular markers that segregated in the BC₁ population placed I1 between the RFLP markers TG20 and TG128 on chromosome 7. The flanking markers were used to verify the assignment of the I1 genotype in the segregating population. The results are discussed with reference to the possibility of cloning Fusarium resistance genes in tomato.     

High-resolution genetic map of the Lv resistance locus in tomato

 Bulked segregant analysis and high-resolution mapping were used to pinpoint the position of the Lv gene for resistance to Leveillula taurica in tomato. Mapping in an F₂, corresponding to more than 3800 gametes, indicates that Lv is positioned within the 0.84-cM interval defined by the RFLP markers CT121 and CT129, with the closest marker, CT121, being only 0.16 cM from the gene. The tight linkage of these markers demonstrates their usefulness in marker-assisted breeding for Lv, and the high-resolution map provides a starting a starting point for positional cloning of this resistance gene. Received: 25 February 1997 / Accepted: 21 March 1997     

Salicylic acid-induced resistance to Fusarium oxysporum f. sp. lycopersici in tomato

We demonstrated that exogenous application of 200 μM salicylic acid through root feeding and foliar spray could induce resistance against Fusarium oxysporum f. sp. Lycopersici (Fol) in tomato. Endogenous accumulation of free salicylic acid in tomato roots was detected by HPLC and identification was confirmed by LC–MS/MS analysis. At 168 h of salicylic acid treatment through roots, the endogenous salicylic acid level in the roots increased to 1477 ng g^?1 FW which was 10 times higher than control plants. Similarly, the salicylic acid content was 1001 ng g^?1 FW at 168 h of treatment by foliar spray, which was 8.7 times higher than control plants. The activities of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and peroxidase (POD, EC 1.11.1.7) were 5.9 and 4.7 times higher, respectively than the control plants at 168 h of salicylic acid feeding through the roots. The increase in PAL and POD activities was 3.7 and 3.3 times higher, respectively at 168 h of salicylic acid treatments through foliar spray than control plants. The salicylic acid-treated tomato plants challenged with Fol exhibited significantly reduced vascular browning and leaf yellowing wilting. The mycelial growth of Fol was not significantly affected by salicylic acid. Significant increase in basal level of salicylic acid in noninoculated plants indicated that tomato root system might have the capacity to assimilate and distribute salicylic acid throughout the plant. The results indicated that the induced resistance observed in tomato against Fol might be a case of salicylic acid-dependent systemic acquired resistance.     

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.     

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.     

Development of a breeder-friendly functional codominant DNA marker for the I2 locus covering resistance to Fusarium oxysporum f. sp. lycopersici

Fusarium oxysporum f. sp. lycopersici Snyder & Hans. (FOL) is a major soil-borne pathogen and the causal agent of Fusarium wilt of tomato, resulting in significant production yield losses. Resistant cultivars have become the most effective method for controlling this fungal disease, and the most important resistance locus to F. oxysporum f. sp. lycopersici in tomato is I2, conferring resistance to race 2 of the pathogen, and widely used in breeding programs. Although this locus was cloned, a robust codominant DNA marker for the I2 locus is not available to date. The development of such a marker has been hindered by the presence of seven homologous sequences at this locus that tend to amplify, and by the absence of information about the structure of the recessive I2 locus. We performed a comparative analysis of the I2 locus nucleotide sequences of tomato genotypes resistant and susceptible to Fusarium wilt. We developed a breeder-friendly functional codominant cleaved amplified polymorphic sequence marker of I2 based on this analysis that can be used in tomato breeding programs for resistance to FOL race 2.     

The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates

Fusarium oxysporum is an asexual fungus that inhabits soils throughout the world. As a species, F. oxysporum can infect a very broad range of plants and cause wilt or root rot disease. Single isolates of F. oxysporum, however, usually infect one or a few plant species only. They have therefore been grouped into formae speciales (f.sp.) based on host specificity. Isolates able to cause tomato wilt (f.sp. lycopersici) do not have a single common ancestor within the F. oxysporum species complex. Here we show that, despite their polyphyletic origin, isolates belonging to f.sp. lycopersici all contain an identical genomic region of at least 8 kb that is absent in other formae speciales and non-pathogenic isolates, and comprises the genes SIX1, SIX2 and SHH1. In addition, SIX3, which lies elsewhere on the same chromosome, is also unique for f.sp. lycopersici. SIX1 encodes a virulence factor towards tomato, and the Six1, Six2 and Six3 proteins are secreted in xylem during colonization of tomato plants. We speculate that these genes may be part of a larger, dispensable region of the genome that confers the ability to cause tomato wilt and has spread among clonal lines of F. oxysporum through horizontal gene transfer. Our findings also have practical implications for the detection and identification of f.sp. lycopersici.     

The in vitro physiological phenotype of tomato resistance to Fusarium oxysporum f. sp. lycopersici

Summary With the aim of dissecting host-parasite interaction processes in the system Lycopersicon aesculentum-Fusarium oxysporum f. sp. lycopersici we have isolated plant cell mutants having single-step alterations in their defense response. A previous analysis of the physiological phenotypes of mutant cell clones suggested that recognition is the crucial event for active defence, and that polysaccharide content, fungal growth inhibition, peroxidase induction in in vitro dual culture and ion leakage induced by cultural filtrates of the pathogen can be markers of resistance. In this paper we present the results of a similar analysis carried out on cell cultures from one susceptible (Red River), one tolerant (UC 105) and three resistant (Davis UC 82, Heinz, UC 90) tomato cultivars. Our data confirm that the differences in the parameters considered are correlated with resistance versus susceptibility in vivo. Therefore, these parameters can be used for early screening in selection programmes. These data, together with those obtained on isolated cell mutants, suggest that the selection in vitro for altered fungal recognition and/or polysaccharide or callose content may lead to in vivo — resistant genotypes. The data are thoroughly discussed with particular attention paid to the importance of polysaccharides in active defense initiation.     

Interactions of the tomato with two formae speciales of Fusarium oxysporum

Tomato cuttings were inoculated with Fusarium oxysporum f.sp. lycopersici (FL) and F. oxysporum f.sp. pisi (FP) by standing the cuttings in suspensions of bud-cells of the fungi. FP never induced external symptoms although the fungus persisted in the lower parts of the cutting. FL at concentrations from 10³ to 10⁶ spores per ml induced typical wilt symptoms but there was subsequent recovery of some cuttings with the production of uninvaded side shoots. When the cuttings were inoculated with mixed suspensions of bud cells of the two fungi there was marked reduction of symptoms. The extent of this reduction was related to the proportion of FP/FL bud cells for a fixed inoculum of FL in the mixture and was moderate at a rate of 1/3 and complete at ratios from 4/1 to 9/1. Mixed suspensions of heat-killed bud cells of FP with live bud cells of FL in the ratio of 4/1 induced normal symptoms and it was concluded that the symptom mitigation induced by FP was related to the presence of living cells of the fungus. Root inoculations with mixed suspensions also gave less wilt than with FL alone. Symptom mitigation was apparently associated with a reduction of the extent of invasion of the cuttings but in vitro tests failed to demonstrate that exudates or extracts from normal or invaded tomato tissue induced any reduction of growth of the tomato pathogen.     

Multiple factors control the level of resistance induced in tomato by Fusarium oxysporum f. sp. cucumerinum

Fourteen wild type and three UV-irradiated isolates of Fusarium oxysporum f. sp. cucumerinum (Foc) were evaluated as to the level of resistance they could induce in tomato to late blight caused by Phytophthora infestans. Tomato plants were induced by applying a suspension of Foc microconidia directly to the surface of the potting media without disturbing the tomato roots. Upper leaves of tomato plants were inoculated with P. infestans, and a reduction in lesion expansion was used as an index of induced resistance. All fourteen wild type isolates of Foc significantly reduced expansion of late blight lesions. One of the wild type isolates produced a significantly weaker resistance response than the other isolates. None of the UV-irradiated isolates induced significant resistance. The same Foc isolates were compared as to their virulence and their pigment production in culture, and considerable variation among them was revealed for both characteristics. Positive correlations existed both between the level of induced resistance and virulence, and between the level of induced resistance and pigmentation. The gradual increment in the level of induced resistance and the exceptions to the correlations between induced resistance and the two characteristics investigated suggest that multiple factors contribute to the induction of resistance by Foc.     

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. Received: 15 July 1997 / Accepted: 1 October 1997     

Induced resistance by the mutualistic endophyte, Fusarium oxysporum strain 162, toward Meloidogyne incognita on tomato

The non-pathogenic endophytic fungus, Fusarium oxysporum strain 162, originally isolated from the endorhiza of tomato roots, reduces damage caused by Meloidogyne incognita, by inhibiting juvenile penetration of and development in the root. However, little is known about the mode of action of this endophyte fungus against the nematode. This study aimed at investigating how the endophyte affects nematode motility and survival and if induced resistance plays a role in the relationship. In a previous study, F. oxysporum strain 162 decreased nematode penetration of tomato up to 60%. In experiments using a split-root chamber to test for induced resistance, nematode penetration, number of galls, and number of egg masses were investigated 2 and 5 weeks after nematode inoculation. Split-root plants treated with F. oxysporum strain 162 showed 26-45% less nematode penetration, 21-36% less galls and a 22-26% reduction in the number of egg masses in the roots not directly inoculated with the fungus when compared to untreated control plants in repeated tests. In conclusion, inoculation of tomato plants with the non-pathogenic fungal endophyte F. oxysporum strain 162 resulted in a signficant reduction of nematode infection, which was in part due to induced resistance in the first 2-3 weeks after fungal inoculation.     

Synthetic chitinase gene driven by root-specific LjNRT2 and AtNRT2.1 promoters confers resistance to Fusarium oxysporum in transgenic tobacco and tomato

Fusarium wilt is a soil-borne disease causing substantial yield losses in various crops and vegetables. We have previously reported the synthetic chitinase (NIC) gene (1.2 kb), in which codon usage of fungus, replaced with that of plant, conferred resistance against Botrytis cinerea. In this study, the NIC or GUS gene was linked to two root-specific promoters, LjNRT2 or AtNRT2.1 (nitrate transporter 2), derived from Lotus japonica and Arabidopsis thaliana, respectively. Transgenic tobacco lines expressing LjNRT2-GUS and LjNRT2-NIC, and tomato lines expressing AtNRT2.1-NIC, were produced by Agrobacterium-mediated transformation. GUS histochemical staining was observed in vascular regions of the roots but was conspicuously absent in the leaves of transgenic plants. Western blot analysis showed the production of NIC proteins in the roots but not in the leaves of transgenic tobacco and tomato lines. These results indicate that LjNRT2 and AtNRT2.1 promoters expressed transgenes in a root-specific manner. When in vitro whole plant resistance assay against Fusarium oxysporum was conducted, transgenic plants showed increased levels of resistance compared to non-transgenic plants. Antifungal activities of the root extract against spore germination of F. oxysporum showed lower CFU (colony-forming unit) than those of the leaf extract. Root colonization assay against F. oxysporum showed much lower CFU values in the roots of transgenic plants than in those of non-transgenic plants. These results suggest that NIC gene triggered by the root-specific promoters successfully expressed only in the roots and conferred increased levels of resistance against the root pathogen, F. oxysporum.     

An isozyme marker for resistance to race 3 of Fusarium oxysporum f. sp. lycopersici in tomato

Summary The levels of erucic acid and other fatty acids in seeds of microspore-derived spontaneous diploid plants from crosses between low and high erucic acid parents were examined. The analysis confirmed that erucic acid is simply inherited and is determined by two genes that act in an additive manner. The effects of the genes for erucic acid on the levels of the other fatty acids was also determined and many significant correlations were found. In particular, erucic acid levels were negatively correlated with oleic acid and linoleic acid levels. The study also illustrates several advantages of using haploidy to analyze the inheritance of agronomically important traits. In particular, the number of phenotypic classes is smaller in androgenic populations and differences between classes are greater than in an F₂ population.     

Microconidia germination of the tomato pathogen Fusarium oxysporum in the presence of root exudates

Abstract

In this study we assessed microconidia germination of the tomato pathogens F. oxysporum f. sp. lycopersici (Fol) and F. oxysporum f. sp. radicis-lycopersici (Forl) in the presence of root exudates. Tomato root exudates stimulated microconidia germination and the level of stimulation was affected by plant age. Treatment of root exudates with insoluble polyvinylpolypyrrolidone, which binds phenolic compounds, indicated that tomato root exudates contain phenolic compounds inhibitory to F. oxysporum microconidia germination. Our study indicates that tomato root exudates similarly stimulate microconidia germination of both Fol and Forl. However, individual F. oxysporum strains differ in the degree of germination response to the root exudates. Furthermore, root exudates from non-host plants also contain compounds that stimulate microconidia germination of Fol. In general, the effects of root exudates from non-host plants did not differ considerably from those of tomato. The ability of phenolic compounds to inhibit germination of Fol seems not to be plant-specific.     

High-resolution genetic and physical mapping of the region containing the Bs2 resistance gene of pepper

The Bs2 resistance gene of pepper confers resistance against the bacterial pathogen Xanthomonas campestris pv. vesicatoria. As a first step toward isolation of the Bs2 gene, molecular markers tightly linked to the gene were identified by randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analysis of near-isogenic lines. Markers flanking the locus were identified and a high-resolution linkage map of the region was developed. One AFLP marker, A2, was found to cosegregate with the locus, while two others, F1 and B3, flank the locus and are within 0.6 cM. Physical mapping of the A2 and F1 markers indicates that these markers may be within 150 kb of each other. Together, these results indicate that the Bs2 region may be cloned either by chromosome walker or landing. The linked markers were also used to characterize gamma-irradiation-induced mutants at the Bs2 locus. Received: 15 January 1999 / Accepted: 11 May 1999     

A comparison of the genetic and physical size of the streptomycin resistance locus in Pneumococcus.

Summary Polyacrylamide gel electrophoresis of the 30S ribosomal proteins derived from six streptomycin resistant strains indicates that each mutation alters the same ribosomal protein (str-r protein). Preliminary data utilizing SDS gels indicates that thestr-r protein has a molecular weight between 10,000 and 20,000 daltons. No significant differences could be detected between the molecular weight of thestr-r protein when it is derived either from a sensitive or from a resistant strain, including those derived from strains carrying multisite mutations of different genetic size. We have estimated the size of the multisitestr-r mutations to be less than 30 base pairs. Two factor crosses withstr-r markers in the trans position demonstrate recombination frequencies expected of closely linked, intragenic markers although cotransfer frequencies, of these same markers from the cis position, are very low. It is concluded that the cotransfer frequencies represent a marker effect and possible explanations are discussed. A reinterpretation of the genetic map of the pneumococcalstr-r locus is presented.Most of the material is taken from a thesis (ALR) submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Graduate Studies, of the State University of New York, Upstate Medical Center