Indirect interaction between an unspecialized endophytic fungus and a polyphagous moth

Despite their ubiquitous occurrence in natural and managed ecosystems, the role of unspecialized fungal endophytes in mediating insect-plant interactions have been largely ignored compared to clavicipitaceous fungal endophytes of grasses. We conducted laboratory and glasshouse experiments to test the hypothesis that an unspecialized fungal endophyte (Acremonium strictum) restricted to the root system of the host plant is able to mediate the interaction between a polyphagous herbivorous insect (Helicoverpa armigera) and its host plant (tomatoes, Lycopersicum esculentum). Our studies indicated a significant variation in the ability of inoculated and control plants to support the life history stages of the insect. Larvae reared on inoculated plants suffer significant reduction in growth rate, prolonged development times, suppressed moulting, and produced smaller pupae that were more likely to die, and emerged adults were less fecund compared to larvae reared on control plants. In glasshouse bioassays were larvae were allowed to graze freely, on inoculated plants only 20% survived to the final stadium compared to 54.5% on control plants. No significant differences in the amount of foliage consumed were found between inoculated and control plants, suggesting that A. strictum does not act as a feeding deterrent for H. armigera larvae. Larvae fed inoculated plants showed a significant increase in the relative consumption rate, although the approximate digestibility of ingested food, and efficiency with which both ingested food and digested food was converted to insect biomass decreased during the same period. In addition to reduced efficiency of food utilization, surviving larvae on inoculated plants displayed a significant reduction in relative growth rate. We hypothesized that alterations in phytosterol composition mediated by infection of the fungal endophyte, may explain the reduced larval performance on inoculated plants.Obwohl unspezialisierte endophytische Pilze in allen Ökosystemen weit verbreitet und häufig sind, blieb ihre Rolle bei Interaktionen zwischen Pflanzen und herbivoren Insekten bisher weitgehend unberücksichtigt. In Labor- und Gewächshausexperimenten haben wir die Hypothese untersucht, dass ein unspezialisierter Wurzelendophyt (Acremonium strictum) die Wechselwirkungen zwischen einem polyphagen Insekt (Helicoverpa armigera) und Tomatenpflanzen (Lycopersicum esculentum) beeinflussen kann. Die Entwicklung der Insekten weist deutliche Unterschiede zwischen inokulierten und Kontrollpflanzen auf. Larven, die auf inokulierten Pflanzen gehalten wurden, zeigten gegenüber solchen von Kontrollpflanzen jeweils signifikant verringerte Wachstumsraten, verlängerte Entwicklungszeiten, verzögerte Häutungen, und bildeten kleinere Puppen aus, welche eine höhere Mortalität aufwiesen. Die geschlüpften Weibchen hatten zudem eine verringerte Fekundität. In Frassversuchen im Gewächshaus überlebten an inokulierten Pflanzen nur 20% der Larven während an Kontrollpflanzen 54,5% überlebten. Da die gefressene Biomasse in beiden Varianten gleich war, ist eine deterrente Wirkung des Pilzes auszuschließen. Vielmehr deuten signifikante Veränderungen der Fraß- und Verwertungsindices sowie die niedrigeren relativen Wachstumsraten darauf hin, das sich die Nahrungsqualität auf inokulierten Pflanzen verschlechtert hatte. Wir diskutieren die Hypothese, dass der Phytosterolgehalt, welcher durch eine Inokulation der Pflanzen verändert wird, zu den verschlechterten Entwicklungsbedingungen der Larven geführt hat.     

Influence of an endophytic fungus on host plant selection by a polyphagous moth via volatile spectrum changes

Recently the role of micro-organisms as mediators of plant-herbivore interactions has been increasingly acknowledged in ecological research. We investigated the interaction between an unspecialized root fungal endophyte (Acremonium strictum) and the polyphagous moth Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), in greenhouse and laboratory bioassays. Specifically we examined in tomato (Lycopersicon esculentum Mill.) the systemic effects of the endophytic fungus on the host selection behaviour of female moths for oviposition and the volatile profiles of host plants to understand the mechanisms acting in this multi-trophic model system. Both laboratory and field strains of H. armigera moths oviposited more on leaves of A. strictum inoculated plants as compared to endophyte-free plants, both in free flight cages and in tethered moth laboratory experiments; the moth’s preferences were significant between 10 and 18 days after inoculation. The analysis of volatile profiles showed strong quantitative differences between treatments. Endophyte inoculated plants emitted diverse terpenes and sesquiterpenes at significantly lower amounts as compared to endophyte free-plants, except for α-terpinene, which did not differ between the treatments, and trans-β-caryophyllene, which was emitted in significantly higher amounts on inoculated plants. β-Thujene and α-phellandrene accounted for 73.3 and 12.0% of total amounts of volatiles emitted from endophyte-free and inoculated plants, respectively. Our findings demonstrate that A. strictum is able to systemically influence the host selection of H. armigera moths for oviposition; conceivably mediated by the induced changes in volatile emissions (and probably additional biochemical parameters of the host plants, which have not been analysed so far). We argue for a more detailed assessment of micro-organisms invisibly colonizing plants, when studying plant-herbivore or multitrophic interactions.     

Changes in ripening-related processes in tomato conditioned by the alc mutant

Summary The alc mutation affects the ripening and storability of tomato fruit. The alteration of fruit color in alc lines is due to a reduction in total pigment and a reduction in lycopene relative to total carotinoids. Polygalacturonase (PG) activity is reduced to less than 5% of normal, and the isozymes PG2a and PG2b are absent in alc fruit. The level of anti-PG precipitable proteins is also reduced to less than 5% of normal. Total polyA + mRNA is not significantly reduced in ripening alc fruit, but hybridization of polyA + mRNA to different ripening-related cDNA clones showed that specific mRNAs are present at reduced levels in the mutant. Specific mRNA levels were reduced to 10%–80% of normal levels, depending on the cDNA clone used as the probe. PG mRNA was present at 5%–10% of the normal level.All effects of alc on fruit ripening are relived in the line Alcobaca-red, which arose spontaneously from the original alc line, Alcobaca. The Alcobaca-red trait segregates as a single dominant trait at or very near the alc locus, and it is probably the result of a reverse mutation at the alc locus.The chromosomal locations of regions homologous to 5 ripening-related cDNA probes were determined. Regions homologous to 4 of these probes map to chromosomes other than chromosome 10, indicating that the effects of alc are transactive. A cDNA clone for PG was homologous to only one chromosomal region. This region is located on chromosome 10, which is also the chromosome on which alc and nor are located.     

An improvement of tomato protoplast culture for rapid plant regeneration

Tomato mesophyll protoplasts were cultured in TM2 medium containing 5.7 M -naphthaleneacetic acid and 2.4 M benzyladenine and were incubated either in stationary culture or on an orbital shaker at 25–30 strokes per min, in combination with interval addition of fresh medium. The effects of stationary and shaking conditions on the growth of the colonies and their subsequent shoot organogenesis were significantly different. The cultures maintained in stationary condition without adding fresh medium accumulated a thin membranous layer on the medium surface and whitish substance in the medium that seemed to precede cell browning and premature colony death. Mild shaking conditions along with the reduction of colony density by one half by dividing the contents of one dish into two dishes, after adding 2 ml of fresh medium on the 4th day and further addition of fresh medium (0.5 ml) on the 8th day of plating, provided optimal conditions for colony growth and suppressed thin layer and whitish substance accumulation. Ten-day-old colonies raised through this protocol regenerated shoots rapidly (within 19–20 days after initial plating) after transfer to regeneration medium (MS medium with 2.8 M zeatin riboside, 0.06–0.1 M gibberellic acid, 4% sucrose and 1% type VII agarose) directly bypassing the callus phase.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige & Skoog (1962) medium - NAA -naphthaleneacetic acid - SPM stroke per min - GLM General Linear Models - 2,4-D 2,4-dichlorophenoxyacetic acid     

Changes in ligno-suberization of cell walls of tomato hairy roots produced by salt treatment: the relationship with the release of a basic peroxidase

A highly basic peroxidase isoenzyme was shown to be released to the culture medium of tomato (Lycopersicon esculentum) hairy roots grown in Murashige-Skoog (MS) liquid medium when it was supplemented with 100 mM NaCl. In this paper we demonstrate that this enzyme is ionically bound to cell walls and that the release was a consequence of the continuous agitation of the tissue in a high ionic strength medium with salt addition. In order to establish the physiological role of this isoenzyme we partially purified it, and we analysed its kinetic properties as coniferyl alcohol peroxidase. The peroxidase isoenzyme showed a high catalytic efficiency for this substrate, which suggests that it would be associated with the ligno-suberization process. To confirm the involvement of this isoenzyme in that process, we studied the pattern of ligno-suberization of the tissue under different conditions of growth. Our results suggest that this basic peroxidase would be indeed involved in ligno-suberization since its leakage from cell walls, induced by 100 mM NaCl in liquid MS, caused less ligno-suberization of exo and endodermis. On the contrary, more ligno-suberization was seen in cell walls when the hairy roots were grown in a salt-supplemented MS solid medium without contact with it, a condition in which the release of the isoenzyme would be avoided. Thus, through the changes produced by the release of the enzyme from its site of action, we could demonstrate the physiological role of this peroxidase in the processing of root cell walls, being part of control mechanisms of ion and water fluxes through the root.     

Biological control of thrips and whiteflies by a shared predator: Two pests are better than one

We studied the capacity of one species of predator to control two major pests of greenhouse crops, Western flower thrips (Frankliniella occidentalis (Pergande)) and the greenhouse whitefly (Trialeurodes vaporariorum (Westwood)). In such a one-predator–two-prey system, indirect interactions can occur between the two pest species, such as apparent competition and apparent mutualism. Whereas apparent competition is desired because it brings pest levels down, apparent mutualism is not, because it does the opposite. Because apparent competition and apparent mutualism occurs at different time scales, it is important to investigate the effects of a shared natural enemy on biological control on a time scale relevant for crop growth. We evaluated the control efficacy of the predatory mites Amblyseius swirskii (Athias-Henriot) and Euseius ovalis (Evans) in cucumber crops in greenhouse compartments with only thrips, only whiteflies or both herbivorous insects together. Each of the two predators controlled thrips, but A. swirskii reduced thrips densities the most. There was no effect of the presence of whiteflies on thrips densities. Whitefly control by each of the two predators in absence of thrips was not sufficient, yet better with E. ovalis. However, whitefly densities in presence of thrips were reduced dramatically, especially by A. swirskii. The densities of predators were up to 15 times higher in presence of both pests than in the single-pest treatments. Laboratory experiments with A. swirskii suggest that this is due to a higher juvenile survival and developmental rate on a mixed diet. Hence, better control may be achieved not only because of apparent competition, but also through a positive effect of mixed diets on predator population growth. This latter phenomenon deserves more attention in experimental and theoretical work on biological control and apparent competition.     

The in vitro metabolism of [8-14C]benzyladenine by excised organs of tomato plants

The metabolic fate of [8-¹⁴C]benzyladenine applied to the excised organs of tomato (Lycopersicon esculentum Mill. cv. Heinz 1370) was investigated after 2 and 6 h of feeding. Although the roots were the most effective at uptake of the cytokinin the leaves metabolised it the most efficiently. The predominant metabolite in all of the tissues was an unknown compound which did not have a retention time corresponding with any of the standards used. The roots contained the most extensive range of metabolites which included the unknown metabolite and compounds co-eluting with adenine, and the riboside, nucleotide and 9-glucoside of benzyladenine. The 9-glucoside was detected only in the root material. The stem yielded the highest levels of radioactivity at the retention times of benzyladenosine-5-monophosphate and benzyladenosine. The radioactivity associated with these two cytokinins was transient in the leaf extract. This organ ultimately yielded radioactivity only at the retention times of the unknown metabolite and adenine. Since only the roots and leaves contained relatively large peaks of radioactivity at the elution volume of adenine it seems that degradative metabolism was more predominant in these organs than in the stem.Abbreviations Ade adenine - Ado adenosine - BA benzyladenine - BAR benzyladenosine - BA3G 3-glucosylbenzyladenine - BA9G 9-glucosylbenzyladenine - BARMP benzyladenosine monophosphate - HPLC high performance liquid chromatography - MS mass spectrometry     

Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation

Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.     

Effects of genotype,explant orientation,and wounding on shoot regeneration in tomato

Summary Effects of genotype and explant orientation on shoot regeneration from cotyledonary explants of tomato were studied using 10 commercially important cultivars. The explant orientation affected shoot regeneration in all the tested genotypes. Cotyledons placed in abaxial (lower surface facing down) orientation consistently produced better shoot regenerative response and produced greater numbers and taller shoots compared to those inoculated in adaxial (upper surface facing down) orientation. Genotypic variation in terms of shoot regeneration response, number of shoots, and shoot height was apparent. Wounding of cotyledonary explants increased shoot regeneration response. However, shoot height was much lower in shoots regenerated from wounded explants compared to those that originated from intact cotyledons. Shoots produced from wounded cotyledons were abnormal in their form and structure.     

Cloning and characterization of a tomato GTPase-like gene related to yeast and Arabidopsis genes involved in vesicular transport

The reduced translation product of a tomato cDNA derived from a gene expressed in a number of tomato tissues of different developmental stages contained sequence motifs characteristic of the GTPase superfamily of proteins. The sequence was closely related to the Sar1 protein of Saccharomyces cerevisiae, a protein essential for the formation of protein transport vesicles at the endoplasmic reticulum (ER) (A. Nakano and M. Muramatsu, Cell Biol 109 (1989): 2677–2691). From analysis of the GTPase superfamily gene sequences, including the tomato SAR-like gene, it is proposed that the SAR genes comprise a distinct GTPase subfamily, presumably with a common, essential function in vesicular transport.     

Evidence for a revertant at the La locus in regenerating hypocotyl segments in tomato

Summary Leafy and leafless phenotypes were regenerated in vitro from hypocotyl segments of leafless forms (reduced and modified) of the homozygous lanceolate (La) mutant in tomato. Segregation of progeny of leafy regenerates into homozygous. mutant (La La), heterozygote (La La ⁺) and normal (La ⁺ La ⁺) indicates that cells forming the shoot apical meristems undergo a genetic reversion, and that the nutrient medium might be selecting for the heterozygote. Among the progeny of the regenerates is a true breeding, unlobed variant. Leaves of the variant are pinnately compound and the margins are entire. Opposite cotyledons followed in development by two simple leaves before the appearance of a pinnately compound leaf with an occasional lanceolate-shaped leaflet suggests that the unlobed variant is morphologically intermediate between La La ⁺ and La ⁺ La ⁺.     

Agrobacterium-mediated transformation of cultivated tomato with construct carrying the nucleoprotein (N) gene from tomato spotted wilt virus (TSWV)

Significant yield losses in commercial tomato production caused by tomato spotted wilt virus (TSWV) are the reason why we have undertaken studies on resistance to this pathogen. One of the possible sources of resistance can be the incorporation of the nucleoprotein N viral gene by Agrobacterium transformation. The N gene was introduced into three Lycopersicon esculentum forms. Out of the total of 3044 cotyledon explants 14.7% regenerated shoots, but only a few were rooted on medium containing kanamycin. The preliminary analysis indicated that 18 plants are putative transformants.     

Expression studies of plant genes differentially expressed in leaf and root tissues of tomato colonised by the arbuscular mycorrhizal fungus Glomus mosseae

Arbuscular mycorrhizal (AM) fungi are a multifaceted group of mutualistic symbionts that are common to terrestrial ecosystems. The interaction between AM fungi and plant roots is of environmental and agronomic importance. Understanding the molecular changes within the host plant upon AM fungal colonisation is a pre-requisite to a greater understanding of the mechanisms underlying the interaction. Differential mRNA display was conducted on leaf tissue of tomato plants colonised and non-colonised by the AM fungus Glomus mosseae and five putative differentially regulated cDNAs were identified. All cDNAs isolated shared high sequence similarity to known plant genes. Differential screening was initially used to establish whether the cDNAs were differentially expressed. Semi-quantitative RT-PCR was used to establish gene expression patterns for all five clones within leaf and root tissue of mycorrhizal and non-mycorrhizal colonised tomato plants. Differential regulation was observed for all five cDNAs. Down-regulation within the leaf tissue of mycorrhizal plants was observed for 4 out of the 5 cDNAs with an up-regulation observed only for one. Tissue specific regulation was observed for several cDNAs, with down-regulation observed in mycorrhizal leaf tissue and up-regulation observed within mycorrhizal root tissue as compared to non-mycorrhizal tissue.     

Totipotency of tomato protoplasts

Summary An efficient and reliable protocol for tomato protoplast isolation, culture, and plant regeneration has been developed. Fourteen diverse cultivars were tested. Fertile plants were regenerated from all 14 cultivars without any modification in the protocol. Plating efficiency (percentage of the protoplasts that formed minicalli) of up to 50% was achieved. Those mini-calli rapidly regenerated shoots at high frequencies. Regenerated shoots can be easily rooted on a basal medium with the appropriate auxin, and have been set to soil within two months after the isolation of the protoplasts.     

Dicyphus tamaninii as a beneficial insect and pest in tomato crops in Catalonia,Spain

The mirid bugDicyphus tamaninii Wagner, is common in Catalonia in vegetable crops which have not been sprayed with insecticide. To study the feeding habits of this bug in tomato crops, several treated and untreated tomato plots were set up at Cabrils Experimental Station (North of Barcelona) during 1983, 1984 and 1985. The results indicate thatTrialeurodes vaporariorum Westwood populations are greatly affected by the mirid bug when no, or only a few, insecticide sprays are applied. However, the bug heavily damaged tomato fruits in unsprayed plots, where bug numbers were high and whitefly numbers low. It seems that tomato fruit is only preferred when whitefly numbers are extremely low. Similar results were obtained in 1984 in a preliminary experiment using cages to exclude the bug.      

Changes in gene expression during programmed cell death in tomato cell suspensions

To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase I. Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals. Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated. During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold. A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD. This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones. CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified. CTD1 is highly similar to Aux/IAA early-auxin-responsive genes. CTD2 corresponds to the tomato RSI-1 gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death. The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes. The possible role of the various predicted gene products in plant PCD is discussed.     

The distribution and utilization of calcium by two tomato (Lycopersicon esculentum Mill.) lines differing in calcium efficiency when grown under low-Ca stress

Two lines of tomato (Lycopersicon esculentum Mill.) representing extremes in utilization effciency of absorbed Ca were studied to detect internal differences in Ca transport and distribution and factors responsible for strain differences in susceptibility to low Ca-stress. Differences in efficiency of Ca use were expressed as CaER (mg of dry weight produced for each mg of Ca absorbed by the plant).Ca-efficiency in line 113(E) appeared to be associated with a slow continuous movement of absorbed Ca, allowing for continued growth of the shoot apex and upper lamina under Ca-deficiency conditions. In the inefficient line 67(I), in contrast, Ca was rapidly deposited in the lower leaves with little upward movement in the plant after absorption.Fractionation of tissue Ca into various chemical forms suggested that Ca inefficiency also was associated with higher concentrations of insoluble Ca in the shoot tissue. The efficient line, although sustaining growth at lower levels of Ca, was capable of maintaining a higher ratio of soluble to insoluble Ca in all shoot tissues.Calcium was concentrated in the lower plant tissues of the inefficient strain, limiting its availability for continued shoot growth.Autoradiographs of lines fed⁴⁵Ca during the final 8 days of a 24-day experiment suggested that upward movement was sustained in line 113(E), in spite of vastly reduced transpiration rates and a root system characterized by leakage of K ions from the roots back into the solution.     

Isolation of a mRNA encoding a nucleoside diphosphate kinase from tomato that is up-regulated by wounding

A cDNA clone (TAB2) encoding a nucleoside diphosphate (NDP) kinase has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) cDNA library. The clone is 590 bp long and exhibits a high degree of sequence identity with spinach NDP kinases I and II, Pisum sativum NDP kinase I, Arabidopsis thaliana NDP kinase, Drosophila melanogaster NDP kinase, Dictyostelium discoideum NDP kinase and human Nm 23-H1 and Nm23-H2. Northern analysis has revealed that the mRNA encoded by TAB2 is up-regulated in both leaf and stem tissue in response to wounding. The increase is apparent within 1 h of wounding and is not further elevated by application of ethylene. Southern blot analysis indicates that TAB2 is a member of a small gene family.     

Low temperature seed germination of several tomato genotypes

Environmental stresses at particularly vulnerable stages during crop development may severely diminish productivity. At temperature of 10 °C or below cultivated tomato germinate slowly if at all. In this study, seven tomato genotypes bred at the Research Institute of Vegetable Crops were evaluated for germination time at 10 °C. Analysis identified that one genotype which has L. chilense in its pedigree, germinated most rapidly while four other genotypes germinated slower. After 21 days, four out of five of the genotypes resulted in seed germination from 81 to 98 %.