Integrated biological control of bacterial speck and spot of tomato under field conditions using foliar biological control agents and plant growth-promoting rhizobacteria

Integration of foliar bacterial biological control agents and plant growth promoting rhizobacteria (PGPR) was investigated to determine whether biological control of bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, and bacterial spot of tomato, caused by Xanthomonas campestris pv. vesicatoria and Xanthomonas vesicatoria, could be improved. Three foliar biological control agents and two selected PGPR strains were employed in pairwise combinations. The foliar biological control agents had previously demonstrated moderate control of bacterial speck or bacterial spot when applied as foliar sprays. The PGPR strains were selected in this study based on their capacity to induce resistance against bacterial speck when applied as seed and soil treatments in the greenhouse. Field trials were conducted in Alabama, Florida, and California for evaluation of the efficacy in control of bacterial speck and in Alabama and Florida for control of bacterial spot. The foliar biological control agent P. syringae strain Cit7 was the most effective of the three foliar biological control agents, providing significant suppression of bacterial speck in all field trials and bacterial spot in two out of three field trials. When applied as a seed treatment and soil drench, PGPR strain Pseudomonas fluorescens 89B-61 significantly reduced foliar severity of bacterial speck in the field trial in California and in three of six disease ratings in the field trials in Alabama. PGPR strains 89B-61 and Bacillus pumilus SE34 both provided significant suppression of bacterial spot in the two field trials conducted in Alabama. Combined use of foliar biological control agent Cit7 and PGPR strain 89B-61 provided significant control of bacterial speck and spot of tomato in each trial. In one field trial, control was enhanced significantly with combined biological control agents compared to single agent inoculations. These results suggest that some PGPR strains may induce plant resistance under field conditions, providing effective suppression of bacterial speck and spot of tomato, and that there may be some benefit to the integration of rhizosphere-applied PGPR and foliar-applied biological control agents.     

Metabolic changes in tomato fruits and seeds after viral, bacterial and fungal infections

The metabolic changes in tomato fruits and seeds separately infected with cucumber mosaic virus, Pseudomonas syringae pv. tomato or Botrytis cinerea were investigated cytochemically. The changes of peroxidase (E.C. 1.11.1.7) and β-glucosidase (E.C. 3.2.1.21) were investigated biochemically as well. Tomato fruits were involved in the study because of their high economic value. Tomato seeds were investigated since they have been used most extensively as a model system for studying the physiology and biochemistry of seed development. The diseases caused by the pathogens under study are of special importance for yield reduction in tomato. The three pathogens provoked local changes in the activities of enzymes under study that affect the infected pericarp tissues and neighboring seeds. It was established non-specific and specific responses. The non-specific responses of invaded tissues were expressed as a local enhancement of peroxidase activity in both pericarp tissues and seeds as well as a decrease in activities of: i. enzymes taking part in aerobic and anaerobic respiration, ii. hydrolases esterase and acid phosphatase involved in the basic metabolism as well as an enhancement of their activities in neighboring tissues. Furthermore, it was observed an enhancement of α-galactosidase activity in infected area was observed. The specific responses depending on the type of the pathogen consisted in an enhancement of glucose-6-phosphate dehydrogenase activity by virus infection and an increase of β-glucosidase activity by fungal invasion.     

Structure and expression of an inhibitor of fungal polygalacturonases from tomato

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.     

Diversity and antagonistic potential of Bacillus spp. associated to the rhizosphere of tomato for the management of Rhizoctonia solani

Bacillus spp. has emerged as the most effective alternative to synthetic chemical fungicides. To get a better insight in the antagonistic potential of Bacillus strains, rhizospheric soil samples of healthy tomato plants from Indo-gangetic plain regions of India were analysed. A total of 108 Bacillus strains were obtained from preliminary screening. Potent strains identified on the basis of in vitro antagonistic and biochemical assays were subjected to diversity analysis using 16S-rDNA, BOX and ERIC-PCR. Furthermore, the four best performing antagonistic Bacillus strains under in vitro plant growth promotion and antagonistic assay were selected for pot experiment. In field study, Bacillus amyloliquefaciens MB101 and Bacillus subtilis MB14 showed drastic reduction in disease index by 55.7 and 41.74% with significant elevation in fruit yield up to 220 and 184 qha^–1, respectively. The present study was successful in selecting effective Bacillus strains by performing phenotypic and genotypic characterisation of Bacillus strains that can be used as an integral component of integrated disease management of tomato root rot and damping-off.     

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     

Inhibition of Telenomus sphingis an egg parasitoid of Manduca spp. by trichome/2-tridecanone-based host plant resistance in tomato

The resistance of accession PI 134417 of the wild tomato Lycopersicon hirsutum f. glabratum C. H. Mull to Manduca sexta (L.) (Lepidoptera: Spingidae) and Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) is conditioned by the high densities of 2-tridecanone-containing, glandular trichomes associated with the foliage. In laboratory experiments, rates of parasitism of M. sexta eggs by Telenomus sphingis (Ashmead) (Hymenoptera: Scelionidae) were lower among eggs on PI 134417 foliage than among eggs on foliage of the cultivated tomato L. esculentum Mill. (cv. Better Boy). The latter is characterized by a significantly lower density of type VI glandular trichomes than PI 134417 and an absence of 2-tridecanone. Parasitism by T. sphingis was also reduced among eggs on foliage of the F₁ hybrid between PI 134417 and L. esculentum. The hybrid foliage lacks 2-tridecanone but has a density of type VI glandular trichomes that is intermediate between those of PI 134417 and L. esculentum, indicating that elevated densities of type VI glandular trichomes adversely affect T. sphingis. This conclusion was further substantiated by the finding that there were no differences among plant lines in the levels of parasitism of M. sexta eggs when the eggs were on foliage that had been divested of glandular trichomes.In bioassays in which T. sphingis adults or immatures in host eggs were exposed to filter paper treated with 2-tridecanone at rates comparable to those associated with PI 134417 foliage, 2-tridecanone was acutely toxic and caused high levels of mortality. In addition, at high concentrations, 2-tridecanone vapors were repellent to T. sphingis adults. However, when exposed to PI 134417 foliage, few T. sphingis adults were killed.Parasitism of M. sexta eggs was unaffected when the eggs were deposited by moths reared as fifth instar larvae on diet containing 2-tridecanone and/or 2-undecanone at levels comparable to those associated with PI 134417 foliage.     

Plant growth regulator and graft control of axillary bud formation and development in the TO-2 mutant tomato

The torosa-2 tomato mutant is characterized by a strong inhibition of release of axillary shoots, that is not under the control of the main apex and IAA. Microscopic examination indicated that about 70% of leaf axils do not have axillary buds. Of the growth regulators tested, gibberellic acid and cytokinins were able to modify the to-2 phenotype: increasing bud number (GA₃ treated) and developing shoots (both substances). Sequential application of growth regulators demonstrated that bud production was only affected by treatments given between sowing time and 32 days after germination. Grafting experiments indicated that endogenous root factors have no essential role in the lateral branching of the genotypes investigated. The control of axillary bud differentiation and the branching pattern in the to-2 appears to be dependent of a complex mechanism involving gibberellins and cytokinins.     

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.     

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.     

Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomatoes using RAPD and RFLP analyses

The Sw-5 locus confers dominant resistance to tomato spotted wilt virus (TSWV). To map the location and facilitate the identification of markers linked to Sw-5 we developed a pair of near-isogenic lines (NILs) and an F₂ Lycopersicon esculentum x L. pennellii population segregating for resistance to TSWV. DNA from the NILs was analyzed using 748 random 10-mer oligonucleotides to discern linked molecular markers using a random amplified polymorphic DNA (RAPD) approach. One random primer (GAGCACGGGA) was found to produce a RAPD band of about 2200 bp that demonstrates linkage to Sw-5. Data from co-segregation of resistance and restriction fragment length polymorphisms (RFLPs) in a F₂ interspecific population position Sw-5 between the markers CT71 and CT220 near the telomere of the long arm of chromosome 9.     

Growth and osmotic adjustment of two tomato cultivars during and after saline stress

The effect of a short period of saline stress was studied in two phenotypically different cultivars, one of normal fruit-size (L. esculentum cv. New Yorker) and one of cherry fruit-size (L. esculentum var.cerasiforme cv. PE-62). In both cultivars the relative growth rate (RGR) and the leaf area ratio (LAR) decreased following salinisation. The leaf turgor potential (_p) and the osmotic potential at full turgor (_os) decreased to the same extent in both cultivars. However, the contributions of organic and inorganic solutes to the osmotic adjustment was different between cultivars. New Yorker achieved the osmotic adjustment by means of the Cl^– and Na⁺ uptake from the substrate, and by synthesis of organic solutes. In the cherry cultivar organic solutes did not contribute to the osmotic adjustment, instead, their contribution decreased after salinisation. After the salt stress was removed, the water stress disappeared, the content of organic solutes decreased in plants of both cultivars and, therefore, their growth was not retarded by the diversion of resources for the synthesis of organic solutes. However, the toxic effects of the Cl^– and Na⁺ did not disappear after removal of the salt stress, and the net assimilation rate (NAR) and the rate of growth (RGR) did not recover.     

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.     

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.     

Expression of a magainin-type antimicrobial peptide gene (MSI-99) in tomato enhances resistance to bacterial speck disease

MSI-99 is a synthetic analog of magainin II (MII), a small cationic peptide highly inhibitory to a wide spectrum of microbial organisms. Tomato plants were transformed to express a gene encoding the MSI-99 peptide and tested for possible enhancement of resistance to important pathogens of this crop. Thirty-six tomato transformants carrying an MSI-99 expression vector designed to target the peptide into extracellular spaces were obtained by Agrobacterium tumefaciens-mediated transformation. Expression of MSI-99 caused no obvious cytotoxic effects in these plants. In the tests with Pseudomonas syringae pv. tomato (bacterial speck pathogen) at 10⁵ CFU/ml, several MSI-99-expressing lines developed significantly fewer disease symptoms than controls. However, MSI-99-expressing lines were not significantly different from controls in their responses to the fungal pathogen Alternaria solani (early blight) and the oomycete pathogen Phytophthora infestans (late blight). These findings are in accordance with our previous in vitro inhibition tests, which showed that the MSI-99 peptide is more inhibitory against bacteria than against fungi and oomycetes. Additional in vitro inhibition assays showed that MSI-99 loses its antimicrobial activity in the total or extracellular fluids from leaflets of non-transformed tomato plants; however, P. syringae pv. tomato could not multiply in the extracellular fluid from an MSI-99-expressing line. Our results suggest that expression strategies providing continuous high expression of MSI-99 will be necessary to achieve significant enhancement of plant disease resistance.Abbreviations AMP Antimicrobial peptide - CFU Colony forming unit - ECF Extracellular fluid - gus -glucuronidase gene - nptII Neomycin phosphotransferase II - SP Signal peptide - TF Total fluidCommunicated by S. Gleddie     

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 %.     

Peroxidase activity and isoenzymes in the culture medium of NaCl adapted tomato suspension cells

The medium of tomato (Lycopersicon esculentum) cells adapted to grow in the presence of 15 g l^–1 NaCl had a higher peroxidase activity than the medium of an unadapted tomato cell line. When the adapted cells were cultured in a medium without NaCl, the value found for peroxidase activity was intermediate. The increase in peroxidase activity was parallel to an increase of lignin-like compounds in the cell walls, as well as to an increased content or appearance of neutral and basic peroxidase isoenzymes. Apparently, the high values of peroxidase activity in the medium of the salt-adapted cells reflect the changed mechanical properties of the cell wall which, in turn, could be related to the salt adaptation process.Abbreviations LO Control tomato cell line unable to grow in the presence of 15 g 1^–1 of NaCl - L15 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in this salt concentration - L15-0 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in the absence of this salt - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PBS phosphate buffer saline     

Biological control of bacterial spot of tomato under field conditions at several locations in North America

Following the relatively successful biological control of bacterial speck of tomato under field conditions at several locations (Phytopathology 92 (2002) 1284), similar selection and testing strategies were employed in an effort to isolate an effective biological control agent for bacterial spot of tomato. Fifty potential biological control agents were isolated from tomato foliage in Alabama (AL) and Florida (FL) and tested under greenhouse conditions in AL for the ability to reduce the foliar severity of bacterial spot of tomato (Lycopersicon esculentum), which is caused by either Xanthomonas campestris pv. vesicatoria or Xanthomonas vesicatoria. Three pseudomonads that provided protection against bacterial speck also were included in the tests. The strains which were most efficacious (i.e., high mean percentage reduction) and consistent (i.e., low standard deviation) in reducing bacterial spot severity in repeated greenhouse experiments were selected for field experiments conducted over the period 1996–1998. Among these strains were Cellulomonas turbata BT1, which provided the highest mean reduction in disease severity [45.2% (SD = 21.0)], and Pseudomonas syringae Cit7 [36.4% (SD = 12.2)], which was the most consistent. Field experiments were conducted in Shorter, AL; Bradenton and Sanford, FL; Clinton, North Carolina; Wooster, Ohio; and London, Ontario, Canada. The highest mean reductions in severity of bacterial spot on foliage, averaged across all locations, were provided by P. syringae Cit7 [28.9% (SD = 11.6)] and Pseudomonas putida B56 [23.1% (SD = 7.4)]. The efficacy and consistency of P. syringae Cit7 against bacterial spot were very similar to those achieved against bacterial speck [28.3% (SD = 12.7)] (Phytopathology 92 (2002) 1284). Unfortunately, neither the bacterial strains nor the standard copper bactericides consistently reduced disease incidence on fruit.     

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.     

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.