Chromosomal localization and molecular-marker tagging of the powdery mildew resistance gene (Lv) in tomato

We report the tagging of a powdery mildew [Leveillula taurica (Lév.) Arnaud.] resistance gene (Lv) in tomato using RAPD and RFLP markers. DNA from a resistant (cv Laurica) and a susceptible cultivar were screened with 300 random primers that were used to amplify DNA of resistant and susceptible plants. Four primers yielded fragments that were unique to the resistant line and linked to the resistance gene in an F₂ population. One of these amplified fragments, OP248, with a molecular weight of 0.7 kb, was subsequently mapped to chromosome 12, 1 cM away from CT134. Using RFLP markers located on chromosome 12, it was shown that approximately one half of chromosome 12 (about 42 cM), in the resistant variety is comprised of foreign DNA, presumably introgressed with the resistance gene from the wild species L. chilense. Further analysis of a backcross population revealed that the Lv gene lies in the 5.5-cM interval between RFLP markers, CT211 and CT219. As a prelude to map-based cloning of the Lv gene, we are currently enriching the density of markers in this region by a combination of RAPD primers and other techniques.     

First record and characterization of a powdery mildew on a member of the Juncaginaceae: <Emphasis Type="Italic">Leveillula taurica</Emphasis> on <Emphasis Type="Italic">Triglochin maritima</Emphasis>

The powdery mildew fungus Leveillula taurica (Erysiphales) is reported for the first time from the monocot Triglochin maritima (Juncaginaceae), a widespread salt marsh plant that causes economic losses because of its high toxicity to young livestock. This is the first report of an erysiphaceous fungus on a member of the Juncaginaceae. Morphological data, obtained by light and scanning electron microscopy, and ITS sequence data provided evidence that this fungus is referable to L. taurica. The ITS sequence for this fungus was identical with those reported for L. taurica hosted by Capsicum annuum in Australia and Elaeagnus angustifolia in Iran. This is the third host species and second monocot, in addition to Allium cepa and Solanum tuberosum, reported for L. taurica from Washington State, where the fungus was unreported before 2004.     

Metabolite analysis for the comparison of irrigated and non-irrigated field grown tomato of varying genotype

Every year the consequences of water deficit on crop yield and quality are profound. The observation that many wild species relatives of cultivated crops display a greater stress tolerance and the fact that the cultivated species generally display only a fraction of the allelic diversity available within the tomato clade suggest that crossing of wild species with elite cultivars could improve the stress physiology of modern crops. To assess this from the basis of chemical composition we applied an established GC-MS based metabolite profiling method to fruits from irrigated and non-irrigated tomato plants either of the cultivated tomato (Solanum lycopersicum) or of its hybrid with its wild species relative (Solanum pennellii). Results are discussed in terms of both the metabolic response to drought stress and the potential of utilizing exotic germplasm as a means to improve agronomically important characteristics of crop species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multiplex PCR to detect four different tomato-infecting pathogens

This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200–800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.     

Loss of Function in Mlo Orthologs Reduces Susceptibility of Pepper and Tomato to Powdery Mildew Disease Caused by Leveillula taurica

Powdery mildew disease caused by Leveillula taurica is a serious fungal threat to greenhouse tomato and pepper production. In contrast to most powdery mildew species which are epiphytic, L. taurica is an endophytic fungus colonizing the mesophyll tissues of the leaf. In barley, Arabidopsis, tomato and pea, the correct functioning of specific homologues of the plant Mlo gene family has been found to be required for pathogenesis of epiphytic powdery mildew fungi. The aim of this study was to investigate the involvement of the Mlo genes in susceptibility to the endophytic fungus L. taurica. In tomato (Solanum lycopersicum), a loss-of-function mutation in the SlMlo1 gene results in resistance to powdery mildew disease caused by Oidium neolycopersici. When the tomato Slmlo1 mutant was inoculated with L. taurica in this study, it proved to be less susceptible compared to the control, S. lycopersicum cv. Moneymaker. Further, overexpression of SlMlo1 in the tomato Slmlo1 mutant enhanced susceptibility to L. taurica. In pepper, the CaMlo2 gene was isolated by applying a homology-based cloning approach. Compared to the previously identified CaMlo1 gene, the CaMlo2 gene is more similar to SlMlo1 as shown by phylogenetic analysis, and the expression of CaMlo2 is up-regulated at an earlier time point upon L. taurica infection. However, results of virus-induced gene silencing suggest that both CaMlo1 and CaMlo2 may be involved in the susceptibility of pepper to L. taurica. The fact that overexpression of CaMlo2 restored the susceptibility of the tomato Slmlo1 mutant to O. neolycopersici and increased its susceptibility to L. taurica confirmed the role of CaMlo2 acting as a susceptibility factor to different powdery mildews, though the role of CaMlo1 as a co-factor for susceptibility cannot be excluded.     

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     

Water Stress-Induced Changes in the Morphology of the Powdery Mildew, Leveillula taurica (Lèv.) Arn.

Water stress in Capsiucm annuum L., induced by means of polyethylene glycol influenced the length and branching frequency of conidiophores and conidial dimensions of the powdery mildew, Leveillula taurica (Lèv.) Arn. Conidiophore branching frequency and length as well as conidial length and width decreased with decreasing relative water content. The availability of water to host plants seems to have a direct effect on the growth vigour and development of the powdery mildew possibly is suggested that water stress may be imposed on host plants by the dry harmattan season resulting in reduced growth vigour of the powdery mildew. Morphology of powdery mildew developing under low water potential may be altered.     

Small-spored Alternaria spp. (section Alternaria) are common pathogens on wild tomato species

The wild relatives of modern tomato crops are native to South America. These plants occur in habitats as different as the Andes and the Atacama Desert and are, to some degree, all susceptible to fungal pathogens of the genus Alternaria. Alternaria is a large genus. On tomatoes, several species cause early blight, leaf spots and other diseases. We collected Alternaria-like infection lesions from the leaves of eight wild tomato species from Chile and Peru. Using molecular barcoding markers, we characterized the pathogens. The infection lesions were caused predominantly by small-spored species of Alternaria of the section Alternaria, like A. alternata, but also by Stemphylium spp., Alternaria spp. from the section Ulocladioides and other related species. Morphological observations and an infection assay confirmed this. Comparative genetic diversity analyses show a larger diversity in this wild system than in studies of cultivated Solanum species. As A. alternata has been reported to be an increasing problem in cultivated tomatoes, investigating the evolutionary potential of this pathogen is not only interesting to scientists studying wild plant pathosystems. It could also inform crop protection and breeding programs to be aware of potential epidemics caused by species still confined to South America.     

QTLs for resistance to powdery mildew in pepper under natural and artificial infections

Epidemics of powdery mildew due to Leveillula taurica is an increasing problem in pepper production areas, particularly in coastal regions or greenhouse cultivation. The highly resistant genitor 'H3' was submitted to genetic analysis and QTL mapping in order to promote the introgression of its oligogenic resistance into large and sweet-fruited cultivars. The doubled-haploid progeny from the cross 'H3' (resistant) by 'Vania' (susceptible) was tested for resistance under both natural field infection and artificial inoculation tests, and QTL detection was compared for those two methods. Seven genomic regions including additive QTLs and epistatic interactions were detected, explaining altogether the major part of genotypic variance. Two genomic regions were common to both the evaluation methods, whereas other QTLs were method-specific, reflecting the environment dependence of powdery mildew epidemics. Orthologies with tomato genomic regions carrying resistance genes to L. taurica and Oidium lycopersicum were revealed by comparative mapping with pepper. Tight linkages between the detected QTLs and virus resistance or fruit color traits in pepper were also shown, which adds to the agronomic importance of these regions in pepper breeding programs.Communicated by G. Wenzel     

Inoculum production and long-term conservation methods for cucurbits and tomato powdery mildews

The behaviour of cucurbit powdery mildews (Podosphaera xanthii and Golovinomyces cichoracearum) and tomato powdery mildew (Oidium neolycopersici) infesting detached cotyledons of Lagenaria leucantha cv. ‘Minibottle’ was studied in order to develop an easy culture method for pure inoculum production. High spore production was found with a combination of mannitol (0.1 m), sucrose (0.02 m) and agar (8 g l⁻¹) in the cotyledon survival medium. Sporulation on cotyledons and viability of conidia were affected by the age of culture for the three species of powdery mildew tested. The age of cotyledons had also an impact of the spore production. This method was used to produce large amounts of inoculum for P. xanthii, G. cichoracearum and O. neolycopersici and enable the development of other species of powdery mildew like Leveillula taurica. Freezing conidia in liquid nitrogen enabled the long-term conservation of P. xanthii without any loss of virulence. The same method was unsuccessful with G. cichoracearum, and L. taurica and partly successful with O. neolycopersici.     

Resistenz verschiedener Kultur- und Wildtomatenpflanzen (Lycopersicon spp.) gegenüber Botrytis cinerea Pers.

Resistance of different cultivated and wild tomato plants (Lycopersicon spp.) to Botrytis cinerea Pers. 20 provenances of different cultivated and wild tomato plants (Lycopersicon spp.) were screened for resistance to Botrytis cinerea Pers. using an in vitro-leaf necrosis test. The Botrytis resistance decreased with increasing age of the leaves corresponding to their insertion height (relative youth resistance respectively senescence susceptibility). The 6 B. cinerea-isolates used for inoculation differed significantly in virulence. With increasing inoculum age a virulence reduction of the various B. cinerea-isolates occurred. Within the investigated test plant collection 2 wild species –L. columbianum and L. hirsutum– proved to be resistant in each stage of development to all B. cinerea-isolates and additionally showed field resistance.     

Developpement de Leveillula taurica en Fonction des Facteurs Climatiques et Sensibilité de Capsicum annuumà Differents Stades Végétatifs

Influence of environmental factors on the development of L. taurica on pepper genotypes To our knowledge, the effects of certain environmental factors, as well as those of host genotype on the development of Leveillula taurica on pepper have not been extensively investigated. To study effects of the above factors on the development of this parasite, two Capsicum genotypes (‘Yolo Wonder' and ‘line 815’) have been tested and compared under different light and relative humidity conditions. In vitro, optimal light intensity for spore germination was situated between 20 and 80 μE/m²/s, and for the growth of germ tubes it was 20 μE/m²/s. In vivo, under continous darkness during 20 days, the different organs of the hosts were only slightly affected. Under light conditions, during 20 days after inoculation, the highest infection level on leaves was observed at 20 μE/m²/s, (60 % of leaves) while with the cotyledons it was observed between 20–80 μE/m²/s. Effects of relative humidity on the development of this parasite were also studied on ‘Yolo Wonder’ and ‘line 815′. With relative humidity below 50 %, 60 % of ‘Yolo Wonder’ plants and 33 % of ‘line 815’ were infected. Under conditions of saturated relative humidity, the level of infection was the inverse, i. e. 13 and 55 %, respectively. Moreover, ‘Yolo Wonder’ plants were found more or less susceptible to infection with L. taurica during their different development stages. At the cotyledon stage all plants were infected. After this stage, up to flowering, susceptibility depended on the physiological age of the leaves. First leaves (older leaves) were not infected, while young ones near the apex became progressively susceptible.     

The Effect of Ventilation on in vitro Response of Seedlings of the Cultivated Tomato and its Wild Salt-tolerant Relative Lycopersicon pennellii to Salt Stress

Organs or plants grown in vitro do not always exhibit the same responses to salinity as the whole plant of same species grown ex vitro. The response to salinity (100 mM NaCl) of seedlings of the wild tomato species Lycopersicon pennellii acc. Atico (Lpa) and of the cultivated tomato L. esculentum cv. M82 (Lem), the former is known as salt tolerant and the second as relatively salt sensitive under ex vitro conditions, was compared under in vitro conditions with three different ventilation regimes. It was found that under salinity shoots of the wild species accumulated the same or even more dry biomass than the control (roots somewhat less) under all ventilation levels. Growth of shoots and roots of the cultivated species was inhibited under the same conditions especially under the high ventilation. Ventilation reduced some abnormalities of leaf development related to hyperhydricity and consequently ventilated leaves exhibited a more compounded structure, increased area, increased resistance to water loss and stomata functioning. Ventilation increased K⁺, Na⁺ and Cl⁻ accumulation in shoots of both tomato species. This was more pronounced under salinity and in Lpa. This work indicates that differences that characterize whole plants of these species in response to salinity under ex vitro conditions are exhibited also in whole plants grown in vitro under high ventilation. It is suggested that ventilation is needed to evaluate well the response of whole plants to salt stress applied in vitro.     

Cytogenetic analysis of Lycopersicon esculentum (+) Solanum etuberosum somatic hybrids and their androgenetic regenerants

The aim of the study was to characterize genomic relationships among cultivated tomato (Lycopersicon esculentum Mill.) (2n=2x=24) and diploid (2n=2x=24) non-tuberous wild Solanum species (S. etuberosum Lindl.). Using genomic in situ hybridization (GISH) of mitotic and meiotic chromosomes, we analyzed intergeneric somatic hybrids between tomato and S. etuberosum. Of the five somatic hybrids, two plants were amphidiploids (2n=4x=48) mostly forming intragenomic bivalents in their microsporocytes, with a very low frequency of multivalents involving the chromosomes of tomato and S. etuberosum (less than 0.2 per meiocyte). Tomato chromosomes showed preferential elimination during subsequent meiotic divisions of the amphidiploids. Transmission of the parental chromosomes into microspores was also evaluated by GISH analysis of androgenic plants produced by direct embryogenesis from the amphidiploid somatic hybrids. Of the four androgenic regenerants, three were diploids (2n=2x=24 or 2n=2x+1=25) derived from reduced male gametes of the somatic hybrids, and one plant was a hypertetraploid (2n=4x+4=52). GISH revealed that each anther-derived plant had a unique chromosome composition. The prospects for introgression of desirable traits from S. etuberosum into the gene pool of cultivated tomato are discussed. Received: 2 August 2000 / Accepted: 4 December 2000     

Development of interspecific Solanum lycopersicum and screening for Tospovirus resistance

Tospovirus has emerged as a serious viral pathogen for several crops including tomato. The tomato production is being severely affected worldwide by Tospovirus. Some reports have been published about the association of plant virus and development of human disease either by direct or indirect consumption. Resistance to this virus has been identified as good source in wild tomato species (Lycopersicum peruvianum). But the introgression of resistance genes into cultivated tomato lines and the development of interspecific hybrid are hampered due to incompatibility, fertilization barriers and embryo abortion. But this barrier has been broken by applying the embryo rescue methods. This study describes the development of interspecific hybrid tomato plants by highly efficient embryo rescue method and screening for Tospovirus resistance. The interspecific hybrid tomato plants were developed by making a cross between wild tomato species (L. peruvianum) and cultivated tomato (Solanum lycopersicum). The immature embryos were cultured in standardized medium and interspecific hybrids were developed from embryogenic callus. The immature embryos excised from 7 to 35 days old fruits were used for embryo rescue and 31 days old embryos showed very good germination capabilities and produced the highest number of plants. Developed plants were hardened enough and shifted to green house. The hybrid nature of interspecific plants was further confirmed by comparing the morphological characters from their parents. The F1, F2 and F3 plants were found to have varying characters especially for leaf type, color of stem, fruits, size, shapes and they were further screened for virus resistance both in lab and open field followed by Enzyme linked Immunosorbant Assay confirmation. Finally, a total of 11 resistant plants were selected bearing red color fruits with desired shape and size.     

Efficacy of Milsana®, a Formulated Plant Extract from Reynoutria sachalinensis, against Powdery Mildew of Tomato (Leveillula taurica)

The efficacy of Milsana^® VP 1999 and 2000 (a formulated plant extract of Reynoutria sachalinensis), known to induce resistance to powdery mildew on cucumbers, was tested against Leveillula taurica (Lév.) Arn. on greenhouse tomato. In four out of five trials, Milsana^® achieved a disease reduction ranging from 42.2 to 64.6%. In one trial only, its efficacy was exceptionally low (23%). Application rates and disease pressure proved to be the main factors affecting the level of control. Milsana^® was significantly less effective than fungicides (alternated DMIs and penconazole) in situ. In contrast, Milsana^® was equally effective to wettable sulphur indicating that its effect was rather preventive than curative. The level of efficacy achieved by either Milsana^® or fungicides did not result in a significant increase of yield. Laboratory tests showed that Milsana^® (VP 1999) had a direct effect on conidial germination. Whether this effect significantly contributes to its field efficacy, remains to be elucidated. Overall, results indicate that Milsana^® could play an important role in disease management of powdery mildew in organic and low input tomato production.     

Solanum pennellii backcross inbred lines (BILs) link small genomic bins with tomato traits

We present a resource for fine mapping of traits derived from the wild tomato species Solanum pennellii (LA0716). The population of backcross inbred lines (BILs) is composed of 446 lines derived after a few generations of backcrosses of the wild species with cultivated tomato (cultivar M82; LA3475), followed by more than seven generations of self‐pollination. The BILs were genotyped using the 10K SOL‐CAP single nucleotide polymorphism (SNP) ‐Chip, and 3700 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs carry, on average, 2.7 introgressions per line, with a mean introgression length of 11.7 Mbp. Whereas the classic 76 introgression lines (ILs) partitioned the genome into 106 mapping bins, the BILs generated 633 bins, thereby enhancing the mapping resolution of traits derived from the wild species. We demonstrate the power of the BILs for rapid fine mapping of simple and complex traits derived from the wild tomato species.     

Mapping QTLs conferring early blight (Alternaria solani) resistance in a Lycopersicon esculentum×L. hirsutum cross by selective genotyping

Most commercial cultivars of tomato, Lycopersicon esculentum Mill., are susceptible to early blight (EB), a devastating fungal (Alternaria solani Sorauer) disease of tomato in the U.S. and elsewhere in the world. Currently, sanitation, long crop rotation, and routine application of fungicides are the most common disease control measures. Although no source of genetic resistance is known within the cultivated species of tomato, resistant resources have been identified within related wild species. The purpose of this study was to identify and validate quantitative trait loci (QTLs) conferring EB resistance in an accession (PI126445) of the tomato wild species L. hirsutum Humb. and Bonpl. by using a selective genotyping approach. A total of 820 BC₁ plants of a cross between an EB susceptible tomato breeding line (NC84173; maternal and recurrent parent) and PI126445 were grown in a greenhouse. During late seedling stage, plants were inoculated with mixed isolates of A. solani and subsequently evaluated for EB symptoms. The most resistant (75 plants = 9.1%) and most susceptible (80 = 9.8%) plants were selected and subsequently transplanted into a field where natural infestation of EB was severe. Plants were grown to maturity and evaluated for final disease severity. From among the 75 resistant plants, 46 (5.6% of the total) that exhibited the highest resistance, and from among the 80 susceptible plants, 30 (3.7% of the total) that exhibited the highest susceptibility, were selected. The 76 selected plants, representing the two extreme tails of the response distribution, were genotyped for 145 restriction fragment length polymorphism (RFLP) markers and 34 resistance gene analogs (RGAs). A genetic linkage map, spanning approximately 1298 cM of the 12 tomato chromosomes with an average marker distance of 7.3 cM, was constructed. A trait-based marker analysis (TBA), which measures differences in marker allele frequencies between extreme tails of a population, detected seven QTLs for EB resistance, one on each of chromosomes 3, 4, 5, 6, 8, 10 and 11. Of these, all but the QTL on chromosome 3 were contributed from the resistant wild parent, PI126445. The standardized effects of the QTLs ranged from 0.45 to 0.81 phenotypic standard deviations. Four of the seven QTLs were previously identified in a study where different populations and mapping strategy were used. The high level of correspondence between the two studies indicated the reliability of the detected QTLs and their potential use for marker-assisted breeding for EB resistance. The location of several RGAs coincided with locations of EB QTLs or known tomato resistance genes (R genes), suggesting that these RGAs could be associated with disease resistance. Furthermore, similar to that for many R gene families, several RGA loci were identified in clusters, suggesting their potential evolutionary relationship with R genes.     

A Solanum neorickii introgression population providing a powerful complement to the extensively characterized Solanum pennellii population

We present a complementary resource for trait fine‐mapping in tomato to those based on the intra‐specific cross between cultivated tomato and the wild tomato species Solanum pennellii, which have been extensively used for quantitative genetics in tomato over the last 20 years. The current population of backcross inbred lines (BILs) is composed of 107 lines derived after three backcrosses of progeny of the wild species Solanum neorickii (LA2133) and cultivated tomato (cultivar TA209) and is freely available to the scientific community. These S. neorickii BILs were genotyped using the 10K SolCAP single nucleotide polymorphism chip, and 3111 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs harbor on average 4.3 introgressions per line, with a mean introgression length of 34.7 Mbp, allowing partitioning of the genome into 340 bins and thereby facilitating rapid trait mapping. We demonstrate the power of using this resource in comparison with archival data from the S. pennellii resources by carrying out metabolic quantitative trait locus analysis following gas chromatography–mass spectrometry on fruits harvested from the S. neorickii BILs. The metabolic candidate genes phenylalanine ammonia‐lyase and cystathionine gamma‐lyase were then tested and validated in F₂ populations and via agroinfiltration‐based overexpression in order to exemplify the fidelity of this method in identifying the genes that drive tomato metabolic phenotypes.