RESEARCH WORK
Effect of the inoculation with rhizospheric bacteria and Trichoderma in wheat (Triticum aestivum L.)
1C. J. Bécquer, 2G. Lazarovits, 2Laura Nielsen, 1Maribel Quintana, 2Modupe Adesina, 2Laura Quigley, 2I. Lalinand2C. Ibbotson
1 Estación Experimental de Sancti Spíritus, Apdo. 2255, Z. P. 1, C. P. 62200, Sancti Spíritus, Cuba. E-mail: pastossp@enet.cu
2 Southern Crop Protection and Food Research Centre, London, Ontario, Canada (Agriculture and Agri-Food Canada)
ABSTRACT
A trial was conducted in greenhouse to evaluate in wheat the effect of the inoculation with the rhizobacteria Sinorhizobium meliloti, Azospirillum zeae and Azospirillum canadense, and with the fungus Trichoderma harzianum. The experimental design was completely randomized, with four replications and five plants per treatment. A treatment fertilized with NH4NO3(150 ppm/kg of soil), an absolute control and 13 inoculated treatments, were used. The aerial dry weight, root dry weight, stem length, germination and content of leaf chlorophyll were evaluated. The separate application of Trichoderma had a positive effect on stem length (5,58 cm), but its presence in some combinations with rhizospheric bacteria was not positive and at the moment of sowing, or afterwards did not show uniform results in the variables. Nevertheless, the treatment Trichoderma + NRG34-DS2 5d stood out in the root dry weight (0,45 g/plant) and the aerial dry weight (0,58 g/plant); as well as the treatment A2-N7, in the chlorophyll content (40,35 SPADS/plant) and stem length (5,50 cm). It is concluded that the simple application of Trichoderma did not exert a positive effect on most variables, and none of the treatments positively influenced germination. It is recommended to evaluate Trichoderma + NRG34-DS2 5d and A2-N7 in field experiments.
Key words: Azospirillum, fungi, plants, Sinorhizobium.
INTRODUCTION
The current concern about the collateral effects of agrochemicals has caused an increasing interest in learning more about the cooperation among the microbial populations of the rhizosphere and the way in which they could be applied in agriculture (Bareaet al., 2004).
Rhizobia are generally considered as symbiotic microbial associates of legumes, through the formation of nitrogen-fixing nodules (Antoun and Prévost, 2005). However, these bacteria can also produce phytohormones and solubilize organic and inorganic phosphates, which play an important role in plant growth (Antoun et al., 1998).
The species belonging to the Azospirillum genus are also considered plant growth promoters (Zahir et al., 2004). These bacteria, in addition to their dinitrogen-fixing activity, have a significant production of phytohormones, which affect root morphology and, thus, improve nutrient uptake from the soil.
Likewise, the filamentous fungus Trichoderma spp., which exerts an efficacious antagonism with phytopathogens (Howell, 1998), induces defense mechanisms and stimulates plant growth (Harman et al., 2004).
There are previous reports about the advantages of the co-inoculation of plants with rhizospheric bacteria and Trichoderma (Bécquer et al. 2004; Rudresh et al., 2005; Saber et al., 2009; Shaban and El-Bramawy, 2011). Yet, there are still many questions to be answered, such as, for example, the way in which these combinations can be applied depending on the plant type, the soil and the environmental conditions, and which microbial species are the most adequate in such combinations. For such reasons, the objective of this trial was to evaluate the effect of the combined inoculation of Sinorhizobium, Azospirillum and Trichoderma in wheat, under controlled conditions and at different moments, for their future application in agricultural practice.
MATERIALS AND METHODS
Origin of the Azospirillum and Sinorhizobium strains. The commercial strains N7 and DS2, belonging to Azospirillum zeae and Azospirillum canadense, respectively,donated by Agriculture and Agri-Food Canada (London, Ontario, Canada); as well as the commercial strains A2 and NRG34, belonging to Sinorhizobium meliloti donated by Agriculture and Agri-Food Canada (Crops Research and Development Centre, Quebec, Canada) were used.
Origin of the Trichoderma strain.The commercial strain TH-382 (Trichoderma harzianum), donated by Agriculture and Agri-Food Canada (Southern Crop Protection and Food Research Centre, Ontario, Canada), was used.
Wheat variety used and provenance. The wheat variety (Triticum aestivum L.) Pioneer 25R47, donated by A&L Canada Laboratories (Ontario, Canada), and recommended by this institution for the soil type used in the trial (Lazarovits, G., pers. com.), was evaluated.
Experimental procedure
Growth and application of the bacterial strains. The strains grew in solid yeast-mannitol medium (Vincent, 1970) and were re-suspended in liquid yeast-mannitol medium, until achieving a titer of 107-108 CFU/mL. The bacterial inoculant was applied at the moment of sowing (1,0 mL/seed) or afterwards (1,0 mL/plant), with previous dilution of 1:10 in a sterile saline solution of 0,85 % NaCl. Five days after sowing a second inoculation was applied, with the same cell concentration (Lupwayi et al., 2004).
Growth and application of the fungal strain. The fungal inoculant was prepared as conidial suspension, by mixing a culture which had been planted for 10 days in PDA (potato dextrose agar) with distilled water and TWEEN-80 (0,01 %), for which the medium surface was scratched with a sterile glass agitator. The final titer of the suspension was 106-107 conidia/mL (Wolffhechell and Jensen, 1992). The conidial suspensions were inoculated to the plants according to the treatment used, in simple form, or in combination with the bacterial inoculants.
Sowing and evaluation of the experiment. Plastic pots were used containing 0,90 kg of non sterilized soil, which was collected in areas belonging to Agriculture and Agri-Food Canada, Experimental Station Delhi (London, Ontario, Canada) (42º 5' N, 80º 3' W). This soil is classified as Brunisolic Gray Brown Luvisol (Canadian System of Soil Classification) according to the report by Chapman and Putman (1966) and is characterized by the predominance of sand (88 %), with the following chemical composition: N: 0,01 %; OM: 1,0 %; P2O5: 217 ppm; K2O: 101,0 ppm; NaCl: 6,0 ppm; Mg: 115,0 ppm; Ca: 62,0 ppm; pH: 6,0. In each pot nine non-sterilized seeds were sown. The evaluation was conducted three weeks after sowing, according to the recommendations made by Somasegaran and Hoben (1994).
Experimental design and treatments. A completely randomized design, with 15 treatments and four replications, was used:
Five plants were used per replication and the following variables were evaluated: aerial dry weight (g/plant); root dry weight (g/plant); stem length (cm/plant); germination (%) and content of leaf chlorophyll, with a chlorophyll detector MINOLTA SPAD-501 (Rodríguez-Mendoza et al., 1998).
Statistical analysis. A variance analysis was applied and the differences among means were found through Duncan's (1955) comparison test. The percentage data were transformed through arcsin»x + 0,375 (Lerch, 1976). The statistical program Statgraphics plus 5.1 for Windows was used.
RESULTS AND DISCUSSION
Chlorophyll content
Regarding the chlorophyll content (table 1), the fertilized control showed the highest value (43,68 SPADS/plant, p < 0,001), compared with the other treatments. It was followed by the treatment in which the strains A2 and N7 were combined, applied at the moment of sowing (40,35 SPADS/plant), which shared common superscripts with other treatments; but was statistically higher (p < 0,001) than the absolute control (38,48 SPADS/plant), as well as than A2-N7 + Trichoderma 5d. (38,55 SPADS/plant), Trichoderma + A2-DS2 5d. (38,40 SPADS/plant), Trichoderma (38,35 SPADS/plant), NRG34-DS2 (37,55 SPADS/plant) and A2-DS2 + Trichoderma 5d (35,93 SPADS/plant).
The fact that the fertilized treatment showed the highest value in this variable is an indication of the high concentration of N induced by the application of the nitrogen fertilizer , because the chlorophyll content is directly proportional to that of N in the plant (Biswas et al., 2000). The microbial combination A2 (S. meliloti) and N7 (A. zeae), at the moment of sowing, was the most effective. According to Askary et al. (2009), Sinorhizobium is a highly efficient genus, due to its plant growth stimulating effect on wheat, in combination with Azospirillum. In this sense, Bécquer et al. (2012a) reported that the combination of A2 and N7 induced the highest chlorophyll content, in this species.
The presence of Trichoderma in the different combinations was not positive, which could be due to the fact that this fungus, because of its antagonistic character with many bacteria (Harman et al., 2004), discreetly interfered the possible benefit for the use of some of the combined treatments, and exerted a more noticeably depressing effect in three of them (A2-DS2 + Trichoderma 5d., Trichoderma + A2-DS2 5d., and A2-N7 + Trichoderma 5d.). This may have depended on the degree of antifungal resistance of the bacterial species used in the combination.
Aerial dry weight
In the aerial dry weight (table 1 ), independently from the fact that the fertilized control (0,82 g/plant) showed statistically higher values (p < 0,05) than the other treatments, the positive effect of A2-N7 + Trichoderma 5 d (0,58 g/plant) was observed, as well as that of Trichoderma + NRG34-DS2 5d (0,58 g/plant). They, although statistically higher than Trichoderma + A2-DS2 5d, A2-N7, NRG34-DS2 + Trichoderma 5d., NRG34-DS2-Trichoderma, A2-N7-Trichoderma and A2-DS2-Trichoderma, did not differ from the absolute control and from other treatments, such as A2-DS2. These results indicate that the positive effect of such combinations on the increase of plant biomass was masked by the microorganisms present in the soil (non sterile) used in the absolute control, in which the autochthonous bacterial community of the rhizosphere should have played an important role. According to Chelius and Triplett (2000), these rhizobacteria, just like the introduced ones, are associated to the plant roots, due to the influence of the organic compounds from the root exudates. Likewise, the fact that the best treatments of the combined inoculation were statistically equal to those of the absolute control corroborates, in a certain way, the hypothesis that the combinations of bacteria with Trichoderma can be affected by the antagonistic action of the fungus.
Stem length
Regarding the stem length (table 2 ), Trichoderma (5,58 cm), A2-N7 (5,50 cm) and NRG34-DS2 (5,50 cm) were statistically higher (p < 0,001) than the absolute control and Trichoderma + A2-DS2 5d. (5,05 cm), NRG34-DS2 + Trichoderma 5d (5,03 cm), NRG34-DS2-Trichoderma (4,95 cm), Trichoderma + A2-N7 5d. (4,80 cm), A2-N7-Trichoderma (4,73 cm) and A2-DS2-Trichoderma (4,68 cm); and did not differ from the other treatments and the fertilized control (5,18 cm).
Although the treatment inoculated only with Trichoderma showed statistically higher values than most treatments, the other two that followed it in importance were composed only by bacteria. This could constitute a tangible proof of the antagonistic influence of the fungus, which was manifested in certain agronomic variables; although its application, separately, exerted a positive effect on stem length. In this sense, Avis et al. (2008) and Shoresh et al. (2010) reported about the biofertilizer properties of Trichoderma, based on the increase of mineral absorption and solubilization, as well as on the production of plant growth stimulating substances. On the other hand, the antagonism of the fungus with a specific strain can be due to the fact that it performs its bio-controlling action through antibiosis, mycoparasitism and competition for nutrients according to Reino et al. (2008). These authors state that most Trichoderma strains produce toxic volatile and non volatile metabolites which prevent the colonization of other antagonistic microorganisms. These metabolites were detected by Saber et al. (2009), in T. harzianum. It is not discarded that some of these compounds have negatively influenced certain functions of the bacteria that stimulate specific indicators of plant growth, such as the above-described variables. Although Bécquer et al. (2001) found that A. brasilense counteracted T. harzianum in an in vitro study; Bécquer et al. (2013) determined that this same fungus species performed antagonistic activity with A. canadense and A. zeae.
Root dry weight
With regards to the root dry weight (table 2 ), Trichoderma + A2-DS2 5d. was statistically higher (p < 0,001) than: A2-N7-Trichoderma , A2-DS2-Trichoderma, NRG34-DS2, A2-N7, absolute control, fertilized control, NRG34-DS2 + Trichoderma 5d and Trichoderma. It was followed by Trichoderma + NRG34-DS2 5d., which was also statistically higher than the other treatments, with the exception of A2-N7-Trichoderma.
Two combined treatments which did not stand out in the previous variables positively influenced this variable. The evident antagonism among the microorganisms used in this trial was not shown in the root dry weight, maybe because Trichoderma, when using its cellulolytic degradation mechanisms on the host roots (Infante et al., 2009), allowed the entrance of the bacteria that were inoculated five days after the application of the fungus, and could have direct incidence on root development (Sarig et al., 1992).
The fact that the simple application of Trichoderma did not exert any positive effect on the roots is important; although Gravel et al. (2006) and Chacón et al. (2007) observed an increase of the root dry weight in tomato and tobacco, respectively, when inoculating them with Trichoderma. On the other hand, Benítez et al. (2004) considered that the colonization of the roots by the fungus, independently from their genus and species, leads to the production of toxic metabolites (phytoalexins, flavonoids and terpenoids, phenol derivatives and other antimicrobial compounds) by the plant, as a response to fungal invasion. The capacity to survive the action of such compounds should vary according to the strain used (Harman et al., 2004).
Germination
The germination values (table 3) showed little or no positive influence of the inoculated strains. The fertilized control as well as the absolute control was statistically higher than A2-N7, Trichoderma, A2-DS2-Trichoderma, A2-N7-Trichoderma, NRG34-DS2 + Trichoderma 5d; and did not differ from the other treatments.
These results do not coincide with the ones found by Benítez et al. (1998), who state that Trichoderma produces growth stimulating substances which influence the increase of the germination rate in the seeds. Likewise, Arora et al. (2001) reported that siderophore-producing strains of S. meliloti increased the germination in peanut, in the presence of Macrophomina phaseolina. However, Canto-Martin et al. (2004) did not observe differences in the germination of chili pepper seeds inoculated with Azospirillum; just like Torres et al. (2003), in Phaseolus vulgaris seeds, inoculated with A. brasilense, Azotobacter chroococcum and Rhizobium leguminosarum, in simple or combined form. Bécquer et al. (2012b) did not find differences in germination either, in wheat, when applying inoculants formed by Azospirillum and Sinorhizobium. Seemingly, the stimulating or depressing capacity of microorganisms in germination depends not only on their genus and species, but also on the plant type to which seeds belong.
General considerations on the analyzed variables
Although several treatments showed high values in, at least, one of the five variables, Trichoderma + NRG34-DS2 5d and A2-N7 stood out with evident superiority in the chlorophyll content and the stem length (A2-N7); as well as in the aerial dry weight and root dry weight (Trichoderma + NRG34-DS2 5d). This last variable is fundamental, because plant growth depends on the roots. According to Sarig et al. (1992), the changes in the morphology and physiology of the root system are one of the most known effects of bacteria on plant growth. The increase of the number of lateral roots and root hairs causes the increase of the available root surface for absorbing water and nutrients (Bai et al., 2003). Hence the importance of the early application of Trichoderma, followed by a combination of rhizospheric bacteria that colonize the inside of the roots, due to the cellulolytic action of the fungus (Infante et al., 2009). Based on these considerations, the treatment Trichoderma + NRG34-DS2 5d. can contribute the best results in agricultural practice; although the combination of the bacterial strains A2 and N7 in the field experiments derived from this study can also be successful.
It should be stated that the fertilized treatment showed significant differences with regards to the absolute control in the chlorophyll content and the aerial dry weight, unlike the other variables (root dry weight, stem length and germination). In this sense, Díaz et al. (2008) obtained similar results in stem length, in field experiments with sorghum, and so did Bécquer et al. (2012a), in germination. These last authors reported that two wheat varieties showed statistically lower values in the root dry weight, in the case of the fertilized treatment compared with the absolute control.
In the variable root dry weight, the fertilized treatment and the absolute control were statistically lower than some of the treatments indistinctly inoculated with Sinorhizobium, Azospirillum and Trichoderma, which also shows the positive effect of these microorganisms on plant development.
CONCLUSIONS
Although the combination of the rhizospheric bacteria with Trichoderma at the moment of sowing, or afterwards, did not show uniform results in the studied variables, the treatment Trichoderma + NRG34-DS2 5d stood out in the root dry weight and aerial dry weight. The treatment A2-N7 also stood out in the chlorophyll content and stem length. The simple application of Trichoderma did not show a positive effect on most variables, and none of the treatments positively influenced germination.
To evaluate the treatments Trichoderma + NRG34-DS2 5d. and A2-N7 in field experiments with wheat is recommended.
Received: June 13, 2014
Accepted: November 4, 2014
