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Agrociencia vol.43 n.6 México Aug./Sep. 2009
Fruit texture related changes and enhanced shelflife through tomato root inoculation with Bacillus subtilis BEB13BS
Cambios relacionados con textura de frutos y mejoramiento de la vida de anaquel por la inoculación de raíces de tomate con Bacillus subtilis BEB13BS
Hortencia G. MenaViolante1, Adrés CruzHernández2, Octavio ParedesLópez2, Miguel Á. GómezLim3, Víctor OlaldePortugal2*
1 Departamento de Biotecnología. Centro de Interdisciplinario de Investigación para el Desarrollo Integral Regional, IPN. Unidad Michoacán. Justo Sierra # 28, Colonia Centro, CP 59510, Jiquilpan, Michoacán México.
2 Departamento de Biotecnología y Bioquímica. Centro de Investigación y de Estudios Avanzados Campus Guanajuato, Km 9.6 Libramiento Norte, Carretera IrapuatoLeón, Apartado Postal 629, CP 36500 Irapuato, Guanajuato, México. * Author for correspondence: (firstname.lastname@example.org)
3 Departamento de Ingeniería Genética. Centro de Investigación y de Estudios Avanzados Campus Guanajuato, Km 9.6 Libramiento Norte, Carretera IrapuatoLeón, Apartado Postal 629, CP 36500 Irapuato, Guanajuato, México.
Received: February, 2008.
Approved: July, 2009.
Bacillus subtilis BEB13bs, a plant growth promoting rhizobacteria, has improved tomato firmness. Since firmer fruits are be expected to be more resistant to spoilage microorganisms and to have a longer shelflife, root inoculation with this strain could be an alternative to extend tomato shelflife. Thus, fruit texturerelated changes were studied in tomato plants (Lycopersicon esculentum Mill.) inoculated with Bacillus subtilis BEB13bs. Treatments were: 1) noninoculated control treatment (CTL); 2) PGPR inoculated treatment (BS13). Evaluation was made at different ripening stages. Pericarp firmness and activity of poligalacturonase (PG), the major cell wall polyuronide degrading enzyme in tomato, were measured. The expression pattern of ripeningrelated genes was also examined. Pericarp firmness was significantly greater in light red (LR) fruits in the BS13 treatment compared to those in the CTL treatment. PG activity was reduced significantly by the BS13 treatment in LR and red (R) fruits compared to the CTL treatment. The expression pattern of Aco, gene coding for 1aminocyclopropane1carboxylate oxidase (ACO), an enzyme that has a regulatory role in ethylene production during fruit ripening, showed a significant decrease in accumulation of the transcript at the R stage in the BS13 treatment compared to that in the CTL treatment. A shelflife test was performed storing fruits at 2527 °C for 10 d at the end of this trial, and fruits in the BS13 treatment were significantly firmer than those in the CTL treatment. Moreover, the percentage decay (nonacceptable fruits) was significantly lower in the BS13 treatment than in the CTL treatment. Additionally, the BS13 treatment promoted a significant increase in fruit fresh weight, size and yield per plant, compared to the CTL treatment. The present results support the development of an environmentfriendly production tool based on PGPR for improving fruit quality through enhanced firmness and shelflife.
Key words: Lycopersicon esculentum, Aco gene expression, firmness, PG activity, quality, ripening.
Bacillus subtilis BEB13bs, una rizobacteria promotora de crecimiento en plantas, ha mejorado la firmeza del tomate. Dado que frutos más firmes tendrían más resistencia al deterioro por microorganismos y una mayor vida de anaquel, la inoculación de la raíz con esta cepa podría ser una alternativa para extender la vida anaquel del tomate. Asi en el presente trabajo se estudiaron los cambios relacionados con la textura de frutos de tomate (Lycopersicon esculentum Mill.) provenientes de plantas inoculadas con Bacillus subtilis BEB13bs. Los tratamientos fueron: 1) tratamiento testigo sin inocular (CTL); 2) tratamiento inoculado con Bacillus subtilis BEB13bs (BS13). Los frutos fueron evaluados en distintos estadios de maduración. Se midió la firmeza del pericarpio y la actividad de poligalacturonasa (PG), enzima mayoritaria de la pared celular implicada en la degradación de poliurónidos. También se realizó un análisis de expresión de genes relacionados con la maduración. La firmeza del pericarpio de los frutos en estadio rojo claro (LR) fue significativamente mayor en el tratamiento BS13 que aquella de los frutos del tratamiento CTL. El tratamiento BS13 redujo significativamente la actividad de PG de los frutos en los estados LR y rojo (R) comparado con el tratamiento CTL. El patrón de expresión de Aco, gen que codifica para la oxidasa del ácido aminociclopropano carboxílico (ACO), enzima reguladora de la síntesis de etileno durante la maduración, mostró una disminución significativa en la acumulación del transcrito en frutos en el estadio R del tratamiento BS13 comparado con aquél de los frutos del tratamiento CTL. Se realizó una prueba de vida de anaquel almacenando frutos a 2527 °C por 10 d; al final de esta prueba; los frutos del tratamiento BS13 resultaron significativamente más firmes que los del tratamiento CTL. Además, el porcentaje de frutos no aceptables fue significativamente inferior en el tratamiento BS13 que en el tratamiento CTL. Adicionalmente, el tratamiento BS13 promovió un incremento significativo del peso fresco y la longitud del fruto, así como del rendimiento por planta, comparado con el tratamiento CTL. Los resultados apoyan el desarrollo de una alternativa de producción ambientalmente amigable basada en la aplicación de BPCV, para mejorar la calidad de frutos en términos de firmeza y vida de anaquel.
Palabras clave: Lycopersicon esculentum, expresión del gen Aco, firmeza, actividad de PG, calidad, maduración.
Rhizobacteria that exert beneficial effects on plant development are referred to as plant growth..promoting rhizobacteria (PGPR) (Kloepper et al., 1980). PGPR strains promote plant growth by acting as biofertilizers improving nutrient status of host plants (Vessey, 2003), acting as bioprotectants via the suppression of plant disease (Whipps, 2001; Zehnder et al., 2001) or as biostimulants through the production of phytohormones and peptides (Glick et al., 1998; Bashan and deBashan, 2004; JiménezDelgadillo, 2004). According to Glick et al. (1998), ethylene, the gaseous phytohormone that influences plant growth and development, is implicated in the mode of action of some PGPR which produce 1aminocyclopropane1carboxylate (ACC) deaminase, an enzyme which could cleave ACC, the immediate precursor to ethylene in the biosynthetic pathway for this hormone in plants. Ethylene controls fruit ripening (Alexander and Grierson, 2002; Alba et al., 2005), a highly complex process characterized by a series of coordinated biochemical and physiological changes that lead to the development of a soft and edible fruit. Ripening influences sensory attributes of fruit quality, including texture. The main cause that reduces fruit quality is an excessive softening which influences shipping, storage and market value (Botella, 2000; Giovannoni, 2001). Fruit softening includes the expression of genes, such as 1aminocyclopropane1carboxylate synthase gene (ACS) and 1aminocyclopropane1carboxylate oxidase gene (ACO), involved in ethylene biosynthesis (Yang and Hoffman, 1984; Bleecker and Kende, 2000), and the polygalacturonase gene (PG).
A diverse range of biotic and abiotic factors can alter agricultural product quality (Schreiner et al., 2000). Benefic rhizosphere microorganisms could be considered as a preharvest biotic factor that affects fruit and vegetable quality (OlaldePortugal and MenaViolante, 2008). In this sense, MenaViolante and OlaldePortugal (2007) reported the positive effects of a PGPR strain (Bacillus subtilis BEB13bs) on tomato fruit quality, particularly on size and texture.
Based on the above information, the objective of our study was to determine the effect of tomato root inoculation with PGPR on fruit texture related changes (physical, biochemical and molecular).
MATERIAL AND METHODS
A greenhouse experiment was set up as follows: tomato (Lycopersicon esculentum Mill. cv Rio Fuego) was grown from seeds in a mixture of peat and perlite (1:1, v/v), seedlings were transplanted after 21 d separately into 3500 cm3 pots containing 5 kg of a mixture methyl bromide sterilized coarse sand and sandy loam soil (1:1 v/v). Bacillus subtilis BEB13bs strain was grown for 12 h at 28 °C in modified potatodextroseagar medium (MPDA) in which agar was not added (Johnson and Curl, 1972). Bacterial suspension was adjusted to yield 1 × 107 CFU mL1 using MPDA. Either 3 mL of MPDA medium (CTL treatment; control) or 3 mL of bacteria suspension (BS13 treatment) were applied into 8 cm deep holes made in pots for transplanting. Plants were irrigated as needed and fertilized weekly with 300 mL per pot of Long Ashton Solution (Hewitt, 1966). There were ten plants per treatment.
Fruits were harvested at different stages of ripeness: green (MG), breaker (B), pink (P), light red (LR) and red (R) (USDA, 1991).
Quality evaluation: size and firmness
Size was determined by measuring fruit fresh weight, size and diameter. All red fruits per plant were sampled and the average weight, length and diameter were calculated. Each plant was a single replication and there were 4 replications per treatment (n=4).
For pericarp tissue, firmness determination, fruits were harvested at B, LR and R stages. Discs were cut from the outer pericarp equatorial wall using a 15mm cork borer. The discs were trimmed to a thickness of 5 mm by excising the peel and then compressed to 3 mm through the short axis. Compression was accomplished with a 7.6cm diameter plate probe attached to a Texture Analyzer TAXT2 (Stable Micro Systems). Crosshead speed was 20 cm min1. Peak heights over the path course were recorded and data were expressed as the reciprocal of N required for the standard compression. Tomato pericarp discs of two opposite points along the equatorial plane of each fruit were evaluated and the average force was recorded (Ahrens and Huber, 1990). Ten fruits from each of the treatments and ripening stages were measured (n=10).
Ten red fruits per treatment were harvested and stored at 2527 °C for 10 d. Fruits were examined every 3 d to determine changes in external quality appearance. Fruits were considered decayed when spots of 1 mm in diameter appeared on their surface due to the natural incidence of spoilage microorganisms. Additionally, a whole fruit firmness evaluation was made at the end of the shelflife test with a 15 mm conical probe using a Texture Analyzer TAXT2 (Stable Micro Systems), measuring the force when loading at 1 mm s1 to a specified distance of 15 mm on two opposite points along the equatorial plane and the average force per fruit was recorded (MenaViolante and OlaldePortugal, 2007). Each fruit was a single replication and there were 10 replications per treatment (n=10).
PG activity assay
Fruits were harvested at G, B, P, LR and R ripening stages; they were sectioned, locular content was removed, the pericarp tissue was frozen in liquid N2 and stored at 80 °C. Protein extraction was performed as described by CarrilloLópez et al. (2002). Protein content was determined by the Bradford method (1976) using bovine serum albumin as standard. The PG activity was assayed by the cyanoacetamide spectrophotometric method (Gross, 1982). Released reducing sugars were quantified using a standard calibration curve obtained with galacturonic acid (Aldrich Chem. Co.). There were two replicates per treatment and ripening stage and six fruit per replicate.
RNA extraction and Northern analysis
Fruits from each treatment were harvested at G, P, LR and R ripening stages. They were sectioned, the locular content was removed the pericarp tissue was frozen in liquid N2 and stored at 80 °C. Total RNA isolation was obtained using the reactive CONCERT, following the manufacturer procedure (Invitrogen). Total RNA (10 mg) was separated in 1% agarose gel containing 1.1% formaldehyde, then it was transferred to a Hybond N+membrane (Pharmacia Biosciences) and crosslinked with UV. Blots were hybridized at 55 °C with cDNA probes of Aco (pTOM13) or Pg (pTOM6), using the Rapidhyb buffer (Amersham Biosciences). Probes were labeled with 32P (Rediprime II, Amersham Biosciences). Filters were exposed for 12 h using Kodak Biomax films (Kodak). Band intensities were measured using the Quantity One 1D Analysis software; they were corrected for differences in loading by comparison to ribosomal RNA.
Experimental design and statistical analysis
Experiments were arranged in a completely randomized design with 20 plants per each treatment. Statistical significance of the data was determined using analysis of variance (ANOVA), and means were compared using minimum significant difference (MSD; p<0.05) (FAUANL, 1994).
RESULTS AND DISCUSSION
Plant yield and fruit size were improved as reported by MenaViolante and OlaldePortugal (2007). PGPR improved fruit size in terms of weight, length and diameter (Table 1). BS13 treatment significantly increased fruit fresh weight (19%), as well as fruit length (20%) compared to the CTL treatment, whereas no effect was found on fruit diameter. Plant yield was 28% higher in the BS13 treatment than that found in the CTL treatment (data not shown).
There is evidence that the mode of action of many PGPR is by increasing the availability of nutrients for the plant in the rhizosphere (BarNess et al., 1992; Richardson, 2001; HernándezDíaz and ChaillouxLaffita, 2001). However, in our study plants were supplied with the proper amounts of all minerals, and no effect of PGPR inoculation was found on the concentration of macronutrients in fruit (data not shown). Although improved mineral uptake by plants was suggested as an important contribution of PGPR to the positive effects on commercial crops (Vessey, 2003), to attribute a phenomenon of quality enhancement by PGPR was due to a nutritional effect is an oversimplified point of view. In fact, positive effects on plant growth independent on mineral content of the host plants have been found using Psolubilizing Bacillus sp. isolates (de Freitas et al., 1997).
Superior tomato shelflife due to the PGPR root treatment was reflected in the significant lower decay (%) presented by the BS13 treatment (Table 2).
Both treatments showed decay after 10 d of storage at 2527 °C, but the incidence was 25% lower in the BS13 treatment than that in the CTL treatment (Table 2). At the end of the shelflife test, fruits in the BS13 treatment were 22% firmer than those in the CTL treatment. Although weight loss of tomatoes in the CTL treatment was higher than that in tomatoes from the BS13 treatment, the differences were not significant (data not shown). This is the first report of fruit shelflife enhanced due to PGPR root inoculation. Since weight was not significantly affected by PGPR treatment, those positive effects in fruits might be derived from fruit morphological changes such as thickness of the outer fruit wall, which delay the attack of microorganisms during storage. Moreover, a significant greater whole fruit firmness in tomatoes from PGPRinoculated plants has been reported (MenaViolante and OlaldePortugal, 2007), and those changes might be also related to possible changes in the locule material and carpel morphology.
The importance of measuring tissue rather than whole fruit firmness, when attempting to relate enzyme activity localized in a specific tissue to changes in texture of that tissue, should be emphasized. PG activity is moderately correlated with firmness of the whole fruit, but is highly correlated with pericarp tissue firmness (Ahrens and Huber, 1990); thus, pericarp tissue firmness was measured in our work. Given that fruit softening increases with ripening (Brady, 1987), in our study the pericarp firmness evaluation was made at different ripeness stages.
Fruits in both treatments showed the typical texture changes during ripening (Table 3) reported by Hobson and Grierson (1993) and Barret et al. (1998). The LR fruits in the BS13 treatment showed a significant increase in pericarp firmness compared to that in the CTL treatment (20%). The B and R fruits did not show significant differences in pericarp firmness between treatments. The fact that fruits in the PGPRtreated plants were firmer in LR fruits suggests a role of PGPR in the ripening process. However, more research must be performed in this area to learn about it.
Firmer fruits are expected to be more resistant to spoilage microorganisms attack (Cooper et al., 1998); in this sense, PGPR treatment not only enhanced whole fruit firmness but also reduced the incidence of spoilage in our study. These findings might be related to PG activity. The PG activity of LR and R fruits in the BS13 treatment was significantly lower (15 and 9%), than that of fruits in the CTL treatment (Figure 1).
Our results suggested that PGPR could enhance tomato fruits shelflife through the reduction of PG activity resulting in increased pericarp tissue firmness and increased resistance to fruit pathogen damage (Kramer et al., 1992). As expected, we found that the activity of the PG enzyme became detectable as the fruits initiated color change and increased progressively as ripening proceeded, with peak activity at the red ripe stage (Hobson and Grierson, 1993). Fruits in both treatments showed the same PG activity pattern; however, root inoculation with PGPR promoted a significant decreased of PG activity in fruits at the later ripeness stages (LR and R) when compared to those from noninoculated plants.
Regarding PG expression, we were not able to relate the above results with the PG transcript accumulation. Fruits in both treatments showed the expected PG transcript accumulation appearing at the onset of ripening (DellaPena et al., 1986) achieving high levels in ripe fruit (DellaPenna et al., 1987, Fischer and Bennett 1991). However, BS13 treatment did not show any effect on the level of PG expression (data not shown). Although PG plays a significant role in texture changes during ripening, it is not the only factor influencing softening.
Fruits in the CTL treatment showed the characteristic pattern of ACO transcript accumulation during fruit ripening, with a basal level at the mature green stage, exponentially increasing till reaching a peak and declining thereafter at the latest ripening stages (Nakatsuka et al., 1998). The Aco transcripts accumulated during ripening in both treatments and showed a drop at the R stage (Figure 2). Interestingly, R fruits in the BS13 treatment presented a decrease in accumulation of the Aco transcript compared to those in the CTL treatment.
Changes in the expression pattern of this ripeningrelated gene, added to the changes in PG enzyme activity, point out the influence of PGPR on the ripening process which results in enhanced fruit quality and shelflife. The fact that bacteria which have those effects on fruit are present only in roots, suggested the involvement of signals that somehow influence the ethylene biosynthetic pathway. PGPR produce phytohormones that are believed to be related to their ability to stimulate plant growth (de Salamone et al., 2001; GutierrezMañero et al., 2001; LucasGarcía et al., 2004). Other growthregulating substances (JiménezDelgadillo, 2004) and volatile organic compounds could be involved (Ryu et al., 2004). The exact mechanism implicated in promoting the reported changes requires further investigation.
Plant root inoculation with PGPR influences tomato texture in the latest fruit ripening stages. The whole fruit and pericarp firmness were increased by PGPRroot inoculation. Shelflife improvement seemed to be favored by the reduction of PG activity, the main cell wall enzyme involved in fruit softening. These findings, together with the drop in the ACO transcript accumulation at the red ripening stage due to bacterial inoculation, suggest that PGPR influences somehow the ripening process.
The authors thank Rosalinda Serrato Flores and Luis Jorge Saucedo Arias for technical assistance. Financial support for this study was provided by the National Council of Science and Technology of Mexico (CONACYT).
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