Yield and Quality Enhancement of Marigold Flowers by Inoculation with Bacillus subtilis and Glomus fasciculatum Antonio Ctirdenas Flores AndrCs Adolfo Estrada Luna Victor Olalde Portugal
ABSTRACT. Marigold (Tagetes erecta L.) is one of the most important flower crops in Mexico. Bacillus subtilis strain BEB-13 and Glomus fasciculatum Gerdemann & Trappe were employed to test their effect on marigold flower yield and quality. Marigold cultivar Alcosa was inoculated with Bacillus and/or Glomus at sowing and transplanting time. The number of inflorescences per plant, flower diameter, fresh weight, xanthophyll content and color were evaluated at the end of the crop AndrCs Adolfo Estrada Luna is affiliated with the Departamento de Ingeniena GenCtica, CINVESTAV-IPN Unidad Irapuato (E-mail:
[email protected]). Antonio Cirdenas Flores (E-mail:
[email protected])and Victor Olalde Portugal (E-mail:
[email protected])are affiliated with the Departamento de Biotecnologia y Bioquimica, CINVESTAV-IPN Unidad Irapuato, Irapuato, Gto., C.P. 36500, Mexico. Address correspondence to: Victor Olalde Portugal at the above address (E-mail:
[email protected]). The authors are grateful to the Consejo Nacional de Ciencia y Tecnologia (CONACyT) of Mexico for economical support. They are thankful to Dr. Gerardo Martinez Soto, University of Guanajuato, for providing the spectrophotometerin color assessment and Dr. Alma A. del Villar-Martinez,Laboratorio de Bioquimica de Alimentos from this department for technical support. They also thank Dr. ~ u n Kilpatrick e Simpson Williamson and Dr. Andres Cruz-Hemindez (CINVESTAV-IPNUnidad Irapuato) for reviewing the manuscript. Joumal of Sustainable Agriculture, Vol. 31(1) 2007 Available online at http://jsa.haworthpress.com O 2007 by The Haworth Press, Inc. All rights reserved. doi: 10.1300/J064v31no1-04
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JOURNAL OF SUSTAINABLE AGRICULTURE
production cycle. Bacillus and/or Glomus treated plants produced 1424% more inflorescences than untreated plants. Although, the treated flowers had significantly higher fresh weight than controls, they did not differ in size. Bacillus improved flower clarity and yellow color but not xanthophyll content, and Glomus enhanced xanthophyll content but not color properties. doi: 10.1300/J064v3 1nO1-04 [Article copies available for a fee from The Haworth Document Delivery Service: I-800-HA WORTH. E-mail address: Website: 0 2007 by The Haworth Press, Inc. All rights reserved.]
KEYWORDS. Arbuscular mycorrhizal fungi, flower color, plant growth promoting rhizobacteria, Tagetes erecta, xanthophylls
INTRODUCTION Marigold (Tagetes erecta L.) also known as "flor de muerto" or "cepaxuchil," is an asteraceous plant native to Mexico. Within Mexico the marigold holds great traditional and economic importance since it has long been used in different religious ceremonies or as a source of food dye ( e g , cheese, egg yolk and poultry skin) (Delgado-Vargas and Paredes-Lopez, 1997). The principal marigold pigments are carotenoids, with lutein (a yellowish xanthophyll) being most abundant. Lutein is synthesized by 1-deoxy-xylulose 5-phosphate (DOXP) pathway and reaches 80-90% of the total pigment content (del Villar-Martinez, 2003). However, little attention has been paid to the yield and quality of marigold flowers. Crop yield and quality are affected by microorganisms associated with the rhizosphere (Glick, 1995; JimCnez-Delgadillo, 2004). Two of the most important and beneficial root-interactive microbes are the plant growth promoting rhizobacteria (PGPR) and the arbuscular mycorrhizal fungi (AMF) (Perotto and Bonfante, 1997). There are a number of wellrecognized PGPR genera such as Azospirillum, Pseudomonas and Bacillus (Glick, 1995; Kloepper et al., 1989; Vessey, 2003) and AMF genera Gigaspora and Glomus, which produce arbuscular mycorrhizal symbioses (Smith and Read, 1997). Both types of microorganisms affect nutrient uptake (Smith and Read, 1997; Vessey, 2003) and secondary metabolism (Kapoor et al., 2002; Lazarovits and Nowak, 1997; Maier et al., 2000), and cause changes in root architecture (JimCnez-Delgadillo, 2003), that can influence plant health, growth, yield and product quality (Hernindez and Chailloux, 2001; Jeffries et al., 2003).
Research, Reviews, Practices, Policy and Technology
23
The purpose of this research was to analyze the effect of Bacillus subtilis (PGPR) (JimCnez-Delgadillo, 2004) and Glomus fasciculatum (AMF) on these traits.
MATERIALS AND METHODS Treatments. Marigold cultivar Alcosa seeds (provided by Alcosa Industries, Apaseo el Grande, Mexico), G. fasciculatum and B. subtilis BEB-13 (strains property of our laboratory) were used to cany out two-season independent experiments, the first from April to August 2003 in a completely randomized design with two treatments: (1) uninoculated marigold control and (2) marigold inoculated with Bacillus. The second experiment from September 2003 to January 2004 in a completely randomized bifactorial design involved two inoculation levels with B. subtilis and two levels with G. fasciculatum, resulting in four treatments: (1) uninoculated control, (2) inoculation with Bacillus, (3) inoculation with Glomus and (4) inoculation with both Bacillus and Glomus. Every treatment had 10 replications. The results were analyzed using an analysis of variance and a Least Significant Difference test at the 5% level. Inoculation procedure. Under green house conditions, surface-sterilized marigold seeds (immersed twice for 5 min in 10% commercial sodium hypochlorite and rinsed with sterile distilled water) were sown in nursery beds with sterile germination substrate. The seeds treated with PGPR were inoculated with 3 ml of a 1 x 107 cfu*ml-I B. subtilis suspension (0.1 absorbance at 535 nm). For treatments involving AMF, 200 g of inoculum containing 73 spores per gram was mixed with the germination substrate before sowing. Three weeks later the plantlets were transplanted to 5 1 pots containing pasteurized sand:loam (2:l) mixture. At the transplanting stage a second inoculation was carried out with 5 ml of B. subtilis suspension, and in those treatments involving AMF, 30 g of inoculum was added at half the depth of the pot. The plants were irrigated maintaining suitable soil humidity and supplied with Long-Ashton nutrient solution in the alternate weeks, applying 44 and 22 mg 1-I doses of phosphorus, respectively, to G. fasciculatum uninoculated and inoculated plants. Measurements. Four months after transplanting, marigold flower yield and quality and mycorrhizal colonization were evaluated. For the purpose of evaluating yield, the flowers were divided into three categories: (1) flowers, corresponding to the mature (fully developed) inflorescences
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number, (2) buttons, corresponding to the immature inflorescences (partially developed) and (3) inflorescences, flowers plus buttons.'To evaluate flower quality, four traits were determined: flower diameter, fresh weight, color and xanthophyll content. Color was measured with the Hunter L (clarity) a (green-red) b (blue-yellow) system (HunterLab, 1996) using a spectrophotometer (Minolta CM-508d; Minolta Camera Co., Ltd., Osaka, Japan). Xanthophyll extraction and content determination was carried out by the method of del Villar-Martinez (2005). Briefly, 0.5 mg of powdered tissue was treated with a hexane, ethanol, acetone, toluene (10:6:7:7) mixture and immediately with 5 ml of 40% KOH in 80% methanol. The mixture was light-protected and shaken at 200 rpm for 16 h at 4°C. Then, 7.5 ml of hexane was added to the mixture under darkness followed by 10% Na2S04to reach a volume of 25 ml and the mixture was left to settle for 1 hour. Finally, the organic phase was recovered and passed through a Na2S04column. The xanthophyll content was determined reading the absorbance at 474 nm using a spectrophotometer (Cary 50 Conc; Varian, Inc., Melbourne, Australia). The root samples were carefully washed, sectioned, stained with trypan blue (Phillips and Hayman, 1970) and examined microscopically to find out the root colonization percentage. Because inflorescence fresh weight markedly decreased through time, these were evaluated in two harvesting times.
RESULTS Compared with uninoculated controls B. subtilis and/or G. fasciculatum positively affected plant yield. The total inflorescence production was significantly increased, by at least, four inflorescences in either treatment involving plant inoculation (Table 1). However, in the first experiment we observed that plants inoculated with the PGPR significantly produced more mature flowers (23 flowers) than uninoculated controls (15 flowers). With the exception of flower fresh weight, the flower traits showed no significant differences among both tests and both harvesting times; hence we only show representative results from one cutting of the second test. There was no significant difference in diameter between uninoculated controls and inoculated plants, nevertheless, the fresh weight showed a significant increase when plants were inoculated with B. subtilis and/or G. fasciculatum (Table 2). Xanthophylls only showed a significant increase (55%) when plants were inoculated with
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Research, Reviews, Practices, Policy and Technology
TABLE 1. Yield obtained from marigold plants inoculated with Bacillus subtilis and/or Glomus fasciculatum. YieldZ
lnoculants
Buttons
Flowers
Bacillus
11.3
+ 0.67 nsY
+ 0.83 ns
Inflorescences
+ 1.52 ns 22.8 + 2.09 ns
33.7
22.3
+ 1.93a
X
33.0 r 1.57a
Glomus
10.2
Bacillus-Glomus
11.5 2 0.81 ns
19.5
+ 1.84 ns
31.0
Control
10.2 2 0.65 ns
16.8 r 2.06 ns
27.0
+ 1.77ab + 1.57b
'yield means of n = 6 plants. Y ~ e a nfrom s the same column preceding 'ns" are not significantly different from each other. 'Means from the same column with different letters are significantly different from each other by LSD test at a = 0.05.
TABLE 2. Flower diameter and fresh weight response to marigold inoculation with Bacillus subtilis and/or Glomus fasciculatum. Flower traitZ
lnoculants Diameter
Fresh weight
Bacillus
6.60 r 1.39 nsY
13.93 5 0.57aX
+ 0.76 ns + 0.47 ns
Glomus
6.85
Bacillus-Glomus
7.46
Control
6.74 r 0.86 ns
12.88 r 0.13ab 12.67 2 0.32ab 10.1 9
+ 0.37b
'yield means of n = 12 flowers (first and second flower of six plants). Y ~ e a nfrom s the same column preceding 'ns" are not significantly different from each other. 'Means from the same column with different letters are significantly different from each other by LSD test at a = 0.05.
G, fasciculatum alone (Figure I), but with no effect on yellow coloration. Regardless of the xanthophyll concentration, only the flowers from plants inoculated with B. subtilis or B. subtilis and G. fasciculatum improved the intensity of their yellow color ("b" parameter) as they did with the "clarity" ("L" parameter) (Figure 2). The "a" parameter (greenred) showed no significant difference between inoculated plants and uninoculated controls (data not shown). Finally, root colonization of marigold inoculated with G. fasciculatum alone (78.5% hyphae; 62.9% arbuscles) or jointly with B. subtilis (63.7% hyphae; 46.9% arbuscles) was significantly higher than in plants treated exclusively with B. subtilis (34.3% hyphae; 0% arbuscles) or noninoculated plants (14.3% hyphae; 0% arbuscles).
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FIGURE 1. Xanthophyll content of flowers from marigold plants treated with Bacillus subtilis andlor Glomus fasciculatum.
Bacillus
Glomus
BacillusGlomus Treatment
Control
Note: Columns indicate the mean of xanthophyll content of n = 10 flowers (first and second flower of five plants). Columns with different letters are significantly different from each other by LSD test at a = 0.05. Error bars represent standard errors.
DISCUSSION The results presented here demonstrate the growth-promoting capability of B. subtilis BEB-13, whose mode of action involves the secretion of small peptides ACC dearninase activity and synthesis of auxin-like molecules (JimCnez-Delgadillo, 2004). These activities are likely responsible for our findings. Both experiments show that marigold plants increased flower fresh weight when treated with B. subtilis. We also observed that inoculation with B. subtilis stimulated marigold flower production, increasing (plant yield) by 14% (inflorescences per plant) when combined with G. fasciculatum and 24% when applied alone. B. subtilis also affected flowering progression by accelerating flower maturity and producing under the same time period a higher number of mature flowers than control and
Research, Reviews, Practices, Policy and Technology
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FIGURE 2. Flower color properties of marigold plants treated with Bacillus subtilis and/or Glomus fasciculatum.
'b, blue-yellow (-60 to 60 bluest to yellowiest); L. clarity (0 to 100 darkest to lightest). @ Means of n = 12 flowers (first and second flowers of six plants) from Bacillustreatments (treatments with 8. subtilis and 8. subtilis with G. fasciculatum). Means of n = 12 flowers (first and second flowers of six plants) from Bacillus noninoculated treatments (treatment with G. fasciculatum alone and uninoculated control). Columns with different letters are significantly different from each other by LSD test at a = 0.05. Error bars represent standard errors.
G. fasciculatum inoculated plants. Similar results have been reported on tomato. Ownley et al. (2001) employing different combinations of Bacillus amyloliquefaciens, Bacillus pumillus and B. subtilis observed a fruit weight increase and accelerated flowering. However, G. fasciculatum treatments did not accelerate flower development, but did stimulate plant yield since plants significantly produced more inflorescences (22%) than uninoculated controls. These results could be attributed to an improved nutritional status. Nonetheless, in an analogous experiment Aboul-Nasr (1996) found that G. etunicatum, independently from the plant's nutritional basis, increased by 34% the number of flowers produced by T. erecta, and if we consider jointly in our test the application of Long-Ashton solution with the recommended phosphorus compensation (22 and 44 mgl-l, respectively, to G. fasciculatum inoculated and uninoculated plants) the increase in flower number could be owing to a different stimulus such as hormone synthesis (Edriss et al., 1984) or a change in photosynthetic rate (Aboul-Nasr, 1996; Auge et al., 1986; Druge and Schonbeck, 1992).
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JOURNAL OF SUSTAINABLE AGRICULTURE
The yield difference between inoculation treatments and controls becomes more important if we consider together the number of inflorescence and the inflorescence fresh weight. In the case of flower quality, this was improved in different ways. Xanthophyll content was enhanced only if G. fasciculatum was applied alone, since G. fasciculatum and B. subtilis together did not generate any increase. In related research, Walter et al. (2000) reported that G. intraradices (AMF) induces the DOXP pathway of the root, producing an accumulation of apocarotenoids (mycorradicin) and mRNA of DXS and DXR (1-deoxyd-xylulose 5-phosphate synthase and reductase, respectively) two key enzymes in DOXP pathway. Nevertheless, in a previous report Mena-Violante (2001) described an increase in fruit xanthophylls and lycopene (carotenoids synthesized via-DOXP, Lichtenthaler, 1999) by G. fasciculatum in water-stressed Capsicum annuum cultivar Ancho San Luis. When we studied color changes in the flowers, we observed some variations in the inoculated ones. The treated flowers showed to be more yellow and to have less intense red hues than the control ones. This was partially supported by the spectrophotometry results since the treatments with PGPR not only generated flowers with lighter color ("L"parameter) but also produced flowers with a more saturated yellow color ("b" parameter). However, according to the measurements there was no difference in red ("a" parameter) between controls and inoculated plants suggesting that other parameters influenced our perception for red tonalities. The existing methods for color measurement of horticultural products (flowers and fruits) are based on the use of colorimeters, spectrophotometers or color charts (Griesbach and Austin, 2005; Kadzere et al., 2006). However, the colorimeters and spectrophotometers present some advantages over color charts in color deterrnination: They can detect imperceptible color changes to the eye, they are not influenced by the judgment of the observer or other interfering factors and they use a worldwide accepted numerical notation which is convenient for data manipulation, interpretation and reporting (van Eck and Franken, 1995). The aforementioned modifications could represent that inoculation with B. subtilis is an advantage in marigold commercialization since flower color is an essential trait in ornamental plants to achieve consumer satisfaction and commercial success (BenMeir et al., 2002). These quality properties also demonstrate that the color changes did not depend on xanthophyll concentration since G. fasciculatum treated plants altered xanthophyll content but not color, and vice versa, B. subtilis
Research, Reviews, Practices, Policy and Technology
29
treated plants altered flower color properties but not xanthophyll content. Accordingly, to further explain the color changes it would be desirable to study the carotenoid profile of the flowers (Cunningham and Gantt, 2002) as well as performing a histological test of the petals since color is affected by the tissue structure (Mena-Violante, 2001). Our findings confirm that B. subtilis and G. fasciculatum are excellent tools for improving marigold cultivation, and also for the first time, demonstrate that the PGPR B. subtilis (BEB-13) shows significant effects on flower quality, a phenomenon rarely studied in the PGPR background. REFERENCES Aboul-Nasr, A. 1996. Effects of vesicular-arbuscular mycorrhiza on Tagetes erecta and Zinnia elegans. Mycorrhiza 6:61-64. Auge, R. M., Schekel, K. A. and R. L. Wample. 1986. Greater leaf conductance of well-water VA mycorrhizal rose plants is not related to phosphorus nutrition. New Phytol 103: 107- 1 16. Ben-Meir, H., Zuker, A,, Weiss, 0 . and A. Vainstein. 2002. Molecular control of floral pigmentation: Anthocyanins. Pp. 253-272. In A. Vainstein (ed.) Breeding for ornamentals: Classical and molecular approaches. Kluwer Academic Publishers, Netherlands. Cunningham Jr, F. X. and E. Gantt. 2002. Molecular control of floral pigmentation: Carotenoids. Pp. 273-293. In A. Vainstein (ed) Breeding forornamentals: Classical and molecular approaches. Kluwer Academic Publishers, Netherlands. del Villar-Martinez, A. 2003. Caracterizaci6n molecular y expresi6n in vitro de las p y E-licopenociclasas de cempaxtichil (Tagetes erecta). PhD thesis. CINVESTAVIrapuato, Irapuato, Gto, Mex. del Villar-Martinez, A., Garcia-Saucedo, P. A., Carabez-Trejo, A., Cruz-Hernhndez, A. and 0. Paredes-Lopez. 2005. Carotenogenic gene expression and ultrastructural changes during marigold flowering. J Plant Physiol 162:1046-1056. DOI: 10.1016/ j.jplph.2004.12.004 Delgado-Vargas, F. and 0 . Paredes-Lopez. 1997. Effects of enzymatic treatments of marigold flowers on lutein isomeric profiles. J Sci Food Agric 45: 1097-1102. Druge, U. and F. Schonbeck. 1992. Effect of vesicular arbuscular mycorrhizal infection on transpiration, photosynthesis and growth of flax (Linum usitatissimum L.) in relation to cy tokinin levels. J Plant Physiol 14 1:40-48. Edriss, M. H., Davis, R. M. and D. W. Burger. 1984. Influence of mycorrhizal fungi on cytokinin production in sour orange. JAm Soc Hortic Sci 109587-590. Glick, B. R. 1995. The enhancement of plant growth by free-living bacteria. Can J Microbiol 4 1:109- 117. Griesbach, R. J. and S. Austin. 2005. Comparison of the Munsell and Royal Horticultural Society's color charts in describing flower color. Taxon 54:771-773.
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HemBndez, M. I. and M. Chailloux. 2001. La nuuici6n mineral y la bifertilizaci6n en el cultivo del tomate (Licopersicon esculentum Mill.). Temas de Ciencia y Tecnologia 5: 11-27. HunterLab color scale. 1996. HunterLab database (http://www.hunterlab.com/appnotes/ an08-96a.pdf). Jeffries, P., Gianinazzi, S., Perotto, S., Tumau, K. and J. M. Barea. 2003. The conuibution of arbuscular mycorrhizal fungi in sustainable maintenance of plant health and soil fertility. Biol Fert Soils 37: 1- 16. DOI: 10.1007/~00374-002-0546-5 JimBnez-Delgadillo, R. 2003. Cambios en la arquitectura y desarrollo de la raiz de Arabidopsis thaliana por efectos de metabolitos con actividad promotora producidos por Bacillus subtilis. In XI Congreso Nacional de Bioquimica y Biologia Molecular de Plantas-5" Simposio, Mixico-E. U. A. (asbtract) JimBnez-Delgadillo, R. 2004. PCptidos secretados por Bacillus subtilis que modifican la arquitectura de la raiz de Arabidopsis thaliana. PhD thesis. CINVESTAVIrapuato, Irapuato, Gto, Mex. Kadzere, I., Watkins, C. B., Merwin, I. A., Akinnifesi, K. K. and J. D. K. Saka. 2006. Postharvest damage and darkening in fresh fruit of Uapaca kirkiana (Muell. Arg.). Pstharv Biol Techno1 39: 199-203. Kapoor, R., Giri, B. and K. Mukerji. 2002. Mycorrhization of coriander (Coriandrum sativum L.) to enhance the concentration and quality of essential oil. J Sci Food Agric 82:339-342. DOI: 10.1002/jsfa.1039 Kloepper, J., Lifshits, R. and R. Zablotowics. 1989. Free-living bacterial inocula enhancing crop productivity. Trends Biotechnol7:39-43. Lazarovits, G. and J. Nowak. 1997. Rhizobacteria for improvement of plant growth and establishment. HortScience. 32: 188-197. Lichtenthaler, H. 1999. The 1-Deoxy-D-Xylulose-5-Phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mo. Biol 50:47-65. DOI: 1040-25 19/99/0601 -0047 Maier, W., Schmidt, J., Nimtz, M., Wray, V. and D. Strack. 2000. Secondary products in mycorrhizal roots of tobacco and tomato. Phytochemistry 54:473-479. Mena-Violante, H. 2001. Calidad de frutos de C. annum L. cv. Ancho San Luis provenientes de plantas con miconiza bajo dos regimenes de riego. MD thesis. CINVESTAV-Irapuato, Irapuato, Guanajuato, Mex. Ownley, B., Seth, D., Hamilton, C. and M. Dee. 2001. Effects of plant-growth-promoting-rhizobacteria on biomass, flowering, and yield of field tomatoes. University of Tennessee. (http:/lbioengr.ag.utk.edu/Extension/ExtProgNegetable/yearNegInit ReportO1/39effects-of-plant.htm). Perotto, S. and P. Bonfante. 1997. Bacterial associations with mycorrhizal fungi: Close and distant friends in the rhizosphere. Trends Microbiol 5:496-501. Phillips, J. and D. Hayman. 1970. Improved procedure of clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Brit Mycol Soc 55: 159-161. Smith, S. E. and D. J. Read. 1997. Mycorrhizal symbiosis. Academic Press, Great Britain. van Eck, J. W. and A. J. M. Franken. 1995. Colours of florets of several gerbera (Gerberajamesonii Bolis ex Adlam) cultivars measured with a colorimeter. Euphytica 84:49-55.
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Vessey, J. 2003. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 255:57 1-586. D O I : 10.1023lA: 1026037216893
Walter, M., Fester, T. and D. Strack. 2000. Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the "yellow pigment" and other apocarotenoids. Plant J 21571-578. D O I : 10.1046/j.l365-31~.2000.00708.x
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