Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. II. Effect of the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis

We have studied the effect of a change in the endogenous hormone equilibria on the competence of tomato (Lycopersicon esculentum) cells to defend themselves against the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Calluses from cvs Davis and Red River, respectively resistant and susceptible to Fusarium and transgenic for an auxin- or cytokinin-synthesizing gene from Agrobacterium tumefaciens, were used. The integration of Agrobacterium hormone-related genes into susceptible cv Red River can bring the activation of defense processes to a stable competence as assessed by the inhibition of mycelial growth in dual culture and gem-tube elongation of Fusarium conidia, the determination of callose contents, peroxidase induction and ion leakage in the presence of fusaric acid. This is particularly true when the transformation results in a change of phytohormone equilibria towards an higher cytokin in concentration. On the contrary, in resistant cv Davis the inhibition of both fungal growth in dual culture and conidia germination is higher when the hormone balance is modified in favour of the auxins. No significant effect was observed for ion leakage and peroxidase induction, probably because of a constitutive overproduction of cytokinins in Davis cells.     

Selection for virus resistance in tomato exposed to tissue culture procedures

Protocols elaborated with the objective of achieving valuable material for selection procedure of variants with virusresistance traits in tomato genotypes are presented. Preliminary results are demonstrated in the domain of testing for variability in somaclones obtained through indirect adventitous organogenesis initiated on leaf explants of cultivated tomato (Lycopersicon esculentum Mill.). Somaclones were grown in greenhouse conditions and variation of their symptoms upon infection with tomato mosaic (ToMV) or cucumber mosaic (CMV) respectively was observed. Tests for resistance to the local isolates of the above cited viruses were performed using enzyme linked immunosorbent assay and back inoculation onto diagnostic plants. Screening data are presented. Desirable variants were selected from cultivars ‘Moneymaker’, ‘Potentat’ and ‘Rutgers’. Some of the ‘Moneymaker’ somaclones exhibited increased tolerance to cucumber mosaic virus, a few seemed to be even fully resistant though most were susceptible as donor plants. The most favourable somaclonal lines are actually further tested and monitored for changes in horticultural characteristics. The described procedure of searching for resistance trait in specific pathogen-free (SPF) plants regenerated from infected tissue looks promising and thus can serve as aid in attaining appropriate objectives of breeding programme. Additionaly experiments were initiated to obtain somaclones from cultivars ‘Beta’, ‘Krakus’ and Stevens Rodade hybrid via regeneration of isolated protoplasts. To this end the callus stage was obtained from all donors.     

Inheritance of resistance to the two-spotted spider mite from L. pimpinellifolium (Jusl.) Mill. accession ‘TO-937’

The two-spotted spider mite (Tetranychus urticae Koch) is an important pest of tomato (Lycopersicon esculentum Mill.) crops in temperate regions as this spider mite has a very large capacity for population increase and causes severe tomato yield losses. There is no described tomato cultivar fully resistant to this pest, although resistant accessions have been reported within the green-fruited tomato wild species L. pennellii (Corr.) D’Arcy and L. hirsutum Humb. & Bonpl. We observed a L. pimpinellifolium (Jusl.) Mill. accession, ‘TO-937’, which seemed to be completely resistant to mite attacks and we crossed it with the susceptible L. esculentum cultivar. ‘Moneymaker’ to obtain a family of generations consisting of the two parents, the F₁, the F₂, the BC₁ to L. esculentum, and the BC₁ to L. pimpinellifolium. This family was evaluated for mite resistance in a polyethylene greenhouse using an experimental design in 60 small complete blocks distributed along 12 double rows. Each block consisted of five F₂ plants in one row and one plant of each of the two parents, the F₁, the BC₁ to L. esculentum, and the BC₁ to L. pimpinellifolium in the adjacent row. Plants at the 10–15 leaf stage were artificially infested by putting on them two pieces of French bean leaf heavily infested with T. urticae. After two months, evaluations of infestation were made by visual observation of mite nets and leaf damage. Plants that were free of signs of mite reproduction on the top half were considered as resistant, plants with silky nets only on their basal leaves, intermediate, and plants with mite reproduction on both basal and top canopies were scored as susceptible. Dominance for resistance appeared because all the ‘To-937’, BC₁ to L. pimpinellifolium, and F₁ plants were resistant. Not all ‘Moneymaker’ plants behaved as susceptible because 35% of plants were intermediate. In the BC₁ to L. pimpinellifolium and the F₂, most plants were scored as resistant, only 7 % BC₁ and 3 % F₂ plants were intermediate, and a single F₂ plant (0.3 %) was susceptible. With these figures, resistance seemed to be controlled by either four or two genes according to whether segregation in the BC₁ or in the F₂, respectively, were considered. These results could in part be explained because of appearance of negative interplot interference due to the high frequency of resistant genotypes within most of the generations. Therefore, the family was evaluated again but using a different experimental design. In the new experiment, 16 ‘TO-937’, 17 ‘Moneymaker’, 17 F₁, 37 BC₁ to L. pimpinellifolium, 38 BC₁ to L. esculentum, and 125 F₂ plants were included. Each of these test plants was grown besides a susceptible ‘Moneymaker’ auxilliary plant that served to keep mite population high and homogeneous in the greenhouse. Negative interplot interference was avoided with this design and all the ‘TO-937’, F₁, and BC₁ to L. pimpinellifolium plants were resistant, all ‘Moneymaker’ test plants were susceptible, and 52 % BC₁ to L. esculentum and 25 % F₂ plants were susceptible, which fitted very well with the expected for resistance governed by a single dominant gene. The simple inheritance mode found will favour sucessful introgression of mite resistance into commercial tomatoes from the very close relative L. pimpinellifolium.     

Characterization of embryogenic mango cultures selected for resistance to Colletotrichum gloeosporioides culture filtrate and phytotoxin

 Embryogenic nucellar cultures of two polyembryonic mango cultivars, ‘Hindi’ and ‘Carabao’, were selected for resistance to the culture filtrate and phytotoxin of a virulent strain of Colletotrichum gloeosporioides Penz. that was isolated from mango leaves. The cultures were recurrently selected either with progressively increasing concentrations of culture filtrate or by continuous challenge with the same concentration of either culture filtrate phytotoxin. Mycelium growth was inhibited when the pathogen was cocultured with the selected, resistant embryogenic cultures. Conditioned plant growth medium containing macerated resistant embryogenic cultures did not inhibit mycelium growth, confirming that extracellular antifungal compounds were involved in the defense response. Enhanced secretion of chitinase and glucanase was observed in the plant growth medium in which resistant embryogenic cultures and regenerated somatic embryos were grown in comparison with the controls. Received: 6 February 1997 / Accepted: 4 November 1997     

Steroidal glycoalkaloid content of potato,tomato and their somatic hybrids

Summary Analyses of leaves and ‘tubers’ from somatic hybrids of potato and tomato (‘pomato’ with plastids of potato, ‘topato’ with plastids of tomato) produced by fusion of protoplasts from liquid cultures of dihaploid potato and mesophyll of tomato revealed the presence of the two major potato glycoalkaloids (α-solanine and α-chaconine) as well as the tomato glycoalkaloid (αtomatine). The total alkaloid content of leaves was greater than that of ‘tubers’ and similar to levels in the foliage of parent plants. However, glycoalkaloids were more abundant in hybrid ‘tubers’ than in normal potato tubers by a factor of 5–15. In hybrid foliage, approximately 98% of the alkaloid present was of potato origin whereas in ‘tubers’ the reverse was the case, with tomatine comprising 60–70% of the total alkaloid. The similarities in alkaloid content and ratios between the pomato and the topato lines indicate that plastomes do not influence the biosynthesis and distribution of these alkaloids. The results indicate that major secondary metabolites may prove useful for assessing the hybrid nature of such plants.     

A functional EDS1 ortholog is differentially regulated in powdery mildew resistant and susceptible grapevines and complements an Arabidopsis eds1 mutant

Vitis vinifera (grapevine) is the most economically important deciduous fruit crop, but cultivated grapevine varieties lack adequate innate immunity to a range of devastating diseases. To identify genetic resources for grapevine innate immunity and understand pathogen defense pathways in a woody perennial plant, we focus in this study on orthologs of the central Arabidopsis thaliana defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). The family of EDS1-like genes is expanded in grapevine, and members of this family were previously found to be constitutively upregulated in the resistant variety ‘Norton’ of the North American grapevine species Vitis aestivalis, while they were induced by Erysiphe necator, the causal agent of grapevine powdery mildew (PM), in the susceptible V. vinifera variety ‘Cabernet Sauvignon’. Here, we determine the responsiveness of individual EDS1-like genes in grapevine to PM and salicylic acid, and find that EDS1-like paralogs are differentially regulated in ‘Cabernet Sauvignon’, while two are constitutively upregulated in ‘Norton’. Sequencing of VvEDS1 and VaEDS1 cDNA and genomic clones revealed high conservation in the protein-encoding sequence and some divergence of the promoter sequence in the two grapevine varieties. Complementation of the Arabidopsis eds1-1 mutant showed that the EDS1-like gene with highest predicted amino acid sequence similarity to AtEDS1 from either grapevine varieties is a functional ortholog of AtEDS1. Together, our analyses show that differential susceptibility to PM is correlated with differences in EDS1 expression, not differences in EDS1 function, between resistant ‘Norton’ and susceptible ‘Cabernet Sauvignon’.     

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.     

An experimental assessment of the factors influencing Agrobacterium-mediated transformation in tomato

Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pTOK233, which contained the GUS reporter gene and a kanamycin-resistance gene nptII, was employed for optimizing the transformation efficiency evaluated by a GUS gene transient expression level. Eight factors including explant types, explant size and source, the concentration of cytokinin, inoculation time, pH of inoculation and cocultivation media, bacterial concentration, acetosyringone concentration, and cocultivation duration were investigated in detail. This optimized protocol was then adopted to obtain transgenic tomato plants resistant to cucumber mosaic virus (CMV) mediated by Agrobacterium tumefaciens, strain LBA4404, carrying a binary vector pR-ΔGDD containing the kanamy cin-resistance gene and CMV replicase gene with GDD deletion. The presence of the CMV-RNA2 gene was confirmed by genomic DNA Southern blot analysis in all transformants analyzed. Field spray test showed that the transgenic tomato plants were resistant to 100 mg/l kanamycin. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 280–284. The text was submitted by the authors in English.     

b-D-glucosidase activity in pelargonium and poinsettia leaves infected by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Pers. ex Fr.

The involvement of β-D-glucosidase activity in grey mould was studied in two ornamental plant species attacked by Botrytis cinerea. β-D-glucosidase activity in the susceptible pelargonium cultivar ‘Shiva’ gradually increased with the disease development in the leaf spots and their surroundings. The endogenic level of the studied hydrolase in the resistant pelargonium ‘Cascade’ was several times higher than in the susceptible cultivar ‘Shiva’ and in principle underwent no changes after inoculation. The postinfection increase in the activity of β-D-glucosidase noted in the leaves of the susceptible poinsettia cultivar ‘Malibu Red’ was evidently weaker in the intensity, but its tendencies were similar to those of the susceptible pelargonium cultivar. In the leaves of the medium-resistant poinsettia ‘Coco White’ the constitutional level of β-D-glucosidase was 2-3-fold higher in that cultivar than in the susceptible cv. ‘Malibu Red’. In attacked leaves of ‘Coco White’, the enzyme activity continued to increase temporarily until the 3^rd h after inoculation. The process of healthy leaf senescence in both species had no significant influence on the change of the studied enzyme activity which was generally low. A high activity of β-D-glucosidase was also observed in the homogenate prepared from mycelium and in the fungal spores.     

Resistance to Phytophthora fragariae var. fragariae in strawberry: the Rpf2 gene

  Phytophthora fragariae var. fragariae is the causal agent of red stele (red core) root rot in strawberry (Fragaria spp.). The inheritance of resistance to one isolate of this fungus was studied in 12 segregating populations of F.×ananassa derived from crosses between four resistant cultivars (‘Climax’, ‘Redgauntlet’, ‘Siletz’, and ‘Sparkle’) and three susceptible cultivars (‘Blakemore’, ‘Glasa’, and ‘Senga’ Sengana’). The analysis clearly supports the hypothesis of a single segregating dominant resistance gene. It is proposed that this gene be designated Rpf2. Received 12 November 1996 / Accepted: 22 November 1996     

Field evaluation of insect resistance in a wild tomato and its effects on insect parasitoids

Populations ofHelicoverpa (=Heliothis) zea (Boddie),Heliothis virescens (F.),Manduca sexta (L.) andM. quinquemaculata (Haw.) and their egg and larval parasitoids were sampled in field plots of the: insect-resistant wild tomato,Lycopersicon hirsutum f. glabratum C. H. Mull, accession PI 134417; susceptible commercial tomato cultivar ‘Better Boy’; F₁ hybrid; and selected, moderately resistant backcross genotype. Densities ofH. zea andH. virescens eggs and small larvae were higher on resistant genotypes than on susceptible genotypes, but densities of large larvae were similar on all genotypes. Densities ofManduca spp. larvae were too low to permit similar analyses of the effects of plant genotype. Rates of egg parasitism byTrichogramma spp. andTelenomus sphingis (Ashmead) were reduced on insect-resistant genotypes. Rates of parasitism by the larval parasitoidsCampoletis sonorensis (Cameron) andCotesia congregata (Say) were reduced on resistant genotypes. No consistent effects on parasitism rates byCotesia marginiventris (Cresson) were observed and parasitism rates byCardiochiles nigriceps Viereck were unaffected.     

Chitinase and peroxidase activities in sunflower hypocotyls: Effects of BTH and inoculation with <Emphasis Type="Italic">Plasmopara halstedii</Emphasis>

Systemic acquired resistance (SAR) can be induced in plants by incompatible pathogens, pathogen derived extracts, or certain chemicals as benzothiadiazole (BTH). The aim of this work was to compare changes in peroxidase and chitinase activities, enzymes considered as PR-proteins, caused by BTH and the pathogen Plasmopara halstedii. Hypocotyls from susceptible and resistant BTH-treated sunflower seedlings showed increased peroxidase and chitinase activities. Inoculation with P. halstedii increased chitinase and peroxidase activities in inoculated hypocotyls from susceptible but not from resistant sunflower seedlings.     

Molecular mapping of the py-1 gene for resistance to corky root rot (Pyrenochaeta lycopersici) in tomato

 We report the molecular mapping of the py-1 gene for resistance to corky root rot [Pyrenochaeta lycopersici (Schneider and Gerlach)] in tomato using RAPD and RFLP marker analysis. DNA from near-isogenic lines (NILs) of tomato differing in corky root rot resistance was screened with 575 random oligonucleotide primers to detect polymorphic DNAs linked to py-1. Three primers (OPW-04, OPC-02, OPG-19) revealed polymorphisms between the NILs. Twelve resistant and eight susceptible DNA pools derived from segregating F₃ families were used to confirm that the RAPD markers were linked to the py-1 gene. Two of the linked amplified fragments, corresponding to OPW-04 and OPC-02, were subsequently cloned and mapped on the tomato molecular linkage map as RFLPs. These clones were located between TG40 and CT31 on the short arm of chromosome 3. Further analysis with selected RFLP markers showed that 7% (8.8 cM) of chromosome 3 of the resistant line ‘Moboglan’ was introgressed from the L. peruvianum donor parent. Three RFLP markers (TG40, TG324, and TG479) from the introgressed part of chromosome 3 were converted to cleaved amplified polymorphism (CAP) markers for use in a polymerase chain reaction (PCR) assay. These PCR markers will allow rapid large-scale screening of tomato populations for corky root rot resistance. Received: 2 January 1998 / Accepted: 12 January 1998     

Pear scab resistance QTLs via a European pear (Pyrus communis) linkage map

Pear scab caused by Venturia pyrina is an economically important disease throughout the world and can cause severe crop loss in susceptible cultivars. The varying range of susceptibility to pear scab in F1 populations has made it possible to identify quantitative trait loci (QTLs). Ninety-five seedlings derived from the cross ‘Abbè Fétel’ (AF) × ‘Max Red Bartlett’ (MRB) were evaluated for scab resistance in greenhouse tests, with 39% being classified as resistant, 33 as moderately susceptible and 28 as highly susceptible. Amplified fragment length polymorphisms (157) and simple sequence repeats (41) were used to construct two maps, one of 908.1 cM (AF) and the other of 879.8 cM (MRB). The analysis of the resistance data collected made it possible to identify two major QTLs on linkage groups 3 and 7 associated with resistance to V. pyrina. Both QTLs explained 88% of the phenotypic variance and the log of odds values were higher than 10, suggesting the involvement of two major genes in pear scab resistance. L. Pierantoni and L. Dondini have contributed equally to this work.     

Silencing of OPR3 in tomato reveals the role of OPDA in callose deposition during the activation of defense responses against Botrytis cinerea

Cis‐(+)‐12‐oxo‐phytodienoic acid (OPDA) is likely to play signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in Solanum lycopersicum (tomato) plant defense, we have silenced the 12‐oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3‐1 and SiOPR3‐2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in OPDA levels and by the production of JA‐Ile being almost abolished. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition, as its pathogen‐induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3‐1 and SiOPR3‐2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in the basal defense of tomato plants against this necrotrophic pathogen.     

In vitro screening for burrowing nematode, Radopholus citrophilus, tolerance and resistance in commercial Anthurium hybrids

Summary A reliable method to screen Anthurium for burrowing nematode resistance and tolerance in vitro was developed using 17 genetically distinct Anthurium cultivars. Based on nonparametric data analysis, tolerance and resistance were found to be independent traits to be evaluated separately. An effective parameter for tolerance evaluation was ranking of relative leaf retention, whereas an effective parameter for resistance evaluation was the ranking of nematode reproduction, log(Rf+1). A comparison of the ranking of leaf retention with ranking of nematode reproduction clustered the cultivar responses to burrowing nematode infection into four groups: intolerant and resistant, moderately tolerant but susceptible, intolerant and susceptible, and tolerant and susceptible. ‘Ozaki’ was identified as an intolerant reference, ‘Nitta’ as a susceptible reference. ‘Blushing Bride’ was the most tolerant cultivar among those screened, but it may not be an ideal tolerant reference due to its low vigor. Future screening for burrowing nematode-tolerant and-resistant cultivars in Anthurium should include ‘Ozaki’ and ‘Nitta’ as internal controls. Evaluation of resistance should be based on a resistance index obtained by log(Rf of hybrid tested +1) divided by log(Rf of ‘Nitta’ +1); tolerance should be based on ranking of relative leaf retention.