Native Trichoderma harzianum strains from Argentina produce indole-3 acetic acid and phosphorus solubilization, promote growth and control wilt disease on tomato (Solanum lycopersicum L.)

1 Introduction

The increase of global demand for agricultural food products over the last 50 years caused the expansion of cultivated areas and resulted in some adverse environmental impacts. At present, crop production involves chemical supplements to increase yield. As an example, fertilizers based on organic nitrate (urea) having a low assimilation, can be dissolved in water or washed from the soil, resulting in excess of nitrogen that ends up in water places causing eutrophication. In general, phosphorus (P) is also supplied to favor plant growth and nutrient uptake; however more than 80% of the P from fertilizers is not available for plant uptake (Kapri and Tewari, 2010). Pesticides are other inputs used in agriculture, resulting in a high cost for producers and causing serious health problems in humans. Moreover, they can produce drastic effects as well as the development of resistant pests (Naseby et al., 2000). At this stage, the reduction or elimination of synthetic products is highly desirable and most promising strategies should be considered. Fortunately, there are some alternatives to increase agricultural production using the beneficial capacities of microorganisms.

Plant growth promoting microbes (PGPM) could contribute increasing the development of plants directly through the improvement of nutrient availability, producing stimulating compounds and releasing phytohormones (Antoun and Prévost, 2005). According to Whipps (2001) diseases caused by pathogens can be indirectly suppressed.

Overall, new tools based on agents of biological control of diseases could be promising strategies to be used alone or in combination with reduced doses of chemicals (Chet and Inbar, 1994; Harman and Kubicek, 1998).

Trichoderma spp. are free living fungi of soil and rhizosphere, plant symbionts and function as parasites of other fungi. They are among the most studied and marketed fungi that are used as biopesticide and biofertilizer soil amendments. Strains with potential for plant growth and health are often isolated from soils (Harman et al., 2004; Lorito et al., 2004).

Many Trichoderma strains are capable to produce compounds as growth regulators that cause sustainable changes in the metabolism of plants. In this sense, Gravel et al. (2007) reported that T. atroviride produced IAA-related indoles under in vitro conditions after the addition of L-tryptophan, suggesting a possible mechanism to increase fresh weight of tomato shoots and roots on tomato. Contreras-Cornejo et al. (2009) suggested that T. virens and T. atroviride synthesize IAA or some derivatives that increase lateral roots in Arabidopsis plants. However, Hoyos-Carvajal et al. (2009) reported that many strains synthesize IAA, but only a few promote plant growth. Thus, the correlation between IAA synthesis and growth promotion in soil-based systems is not clear yet (Nieto-Jacobo et al., 2017).

Argentina is mainly an agro-exporting country in which horticultural crop region reaches 409,321 ha with an estimated annual yield of around 10,500,000 tons. The tomato (Solanum lycopersicum L.) is one of the most demanded horticultural crops in our country and its production is mainly done in greenhouses (Argerich, 2011). In this context, the objective is to optimize the yield, the soil resources and minimize diseases. To increase tomato production, continuous nutrient supply and repeated chemical control of diseases are usually applied. In this sense, it was found that the south of the Pampas region had low levels of soil P (<10 ppm P-Bray I), and in most cases, a large amount of P added as fertilizer remains retained in the soil due to its high adsorption capacity and not available for the growth of plants (Silva Rossi et al., 2013). Therefore, strategies for the solubilization of P are sought.

Fungal disease is a crucial problem in intensive cultivation and every year the crop losses are caused by fungal species such as Cladosporium fulvum, Fusarium oxysporum, Alternaria dauci f. sp. solani, Phytophthora infestans and capsici (Adlercreutz et al., 2014). Because laws limit the use of chemicals in agriculture, farmers are looking for other strategies for a sustainable production with the maintenance of productivity, and beneficial microorganisms could represent a promising alternative for the achievement of this goal. The soils of the Argentine Pampas have a high microbial diversity, but little has been explored in their potential as Trichoderma hot spots. In this context, our aim was to isolate, characterize and select strains of Trichoderma spp. native from this region with potential as growth promoters and as biocontrol agents for wilt disease caused by F. oxysporum on tomato.

2 Materials and methods

3 Results

3.2

3.2 Dual confrontation assays between Trichoderma and F. oxysporum

All Trichoderma strains showed in vitro antagonistic effect against F. oxysporum and evidenced inhibition of the pathogeńs growth in the range of 20–100%; in addition, twelve strains inhibited mycelial growth of F. oxysporum by more than 50%. The highest biocontrol effect was obtained for FCCT 42, which showed a 100% of biocontrol efficiency. Strains FCCT 58, FCCT 63, FCCT 187-2 and FCCT363-2 reduced the pathogeńs growth by more than 90% (Fig. 1).

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Fig. 1

Inhibition effects of Trichoderma strains over Fusarium oxysporum in dual assay after seven days. Mean averages (n = 3). Different letters on bars indicate statistical differences between treatments according to Tukeýs Test (P ≤ 0.05).

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In the intermingling zone between the pathogen and each isolate, the plasmolysis of pathogen hyphae or coiling by Trichoderma was evidenced.

3.5

3.5 Effects of Trichoderma on plant growth and pathogenicity

3.5.1

3.5.1 Influence of Trichoderma on the promotion of tomato plant growth

Tomato plants inoculated with FCCT 16, FCCT 58, FCCT 199-2 and FCCT 363-2 strains increased plant growth (Fig. 2). SPAD units (indirect chlorophyll content indicator) were higher in plants inoculated with FCCT 58 and FCCT 199-2, being the last strain mentioned which showed the highest leaf area. Although the remaining strains also increased the leaf area and the SPAD reading, no significant differences were detected in comparison with the controls (Table 3).

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Fig. 2

In vivo plant growth promotion assay of tomato plants (Solanum lycopersicum L. var platense). Shoot and their respective root after 45 days post inoculation of seed with strains FCCT 16 (a) FCCT 58, (b) FCCT 199-2 (c), FCCT 363-2 (d). On left control plants, inoculated on right.

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Table 3 Evaluation of the four most potential Trichoderma isolates for their plant growth-promoting potential in tomato under greenhouse conditions.

Treatment	Spad units	% over control	Leaf área (cm)	% over control	Stem length (cm)	% over control	Shoot Fresh Weight (g)	% over control	Shoot Dry Weight (g)	% over control	Root Fresh Weight (g)	% over control	Root Dry Weight (g)	% over control
control	19.37 ± 2.41c		73.52 ± 19.15b		22.44 ± 7.37b		1.25 ± 0.41b		0.23 ± 0.05b		0.52 ± 0.14b		0.03 ± 0.01b
FCCT16	21.36 ± 2.51bc	10.2	137.69 ± 17.72ab	87.3	31.9 ± 2.40a	42.1	4.16 ± 0.94a	332	0.55 ± 0.18a	135.6	1.53 ± 0.77a	194.2	0.12 ± 0.06a	400
FCCT58	29.53 ± 3.11a	52.4	140.19 ± 30.86ab	90.7	29.00 ± 4.09a	29.2	3.09 ± 0.83a	247.2	0.48 ± 0.17a	118.1	0.89 ± 0.41ab	71.10	0.07 ± 0.02ab	233
FCCT 199-2	26.70 ± 3.10ab	37.8	176.17 ± 42.08a	139.6	27.5 ± 7.69a	22.7	3.36 ± 1.08a	268.8	0.55 ± 0.07a	135.5	1.54 ± 0.89a	196.1	0.10 ± 0.02ab	333
FCCT 363-2	25.93 ± 4.00abc	33.8	103.66 ± 42.15b	140.9	29.44 ± 1.35a	31.2	2.83 ± 1.06ab	226.4	0.36 ± 0.25b	53.91	0.78 ± 0.45b	50.0	0.05 ± 0.03ab	166

*Values are means and standard deviation n = 3 replications and data calculated per plant after 45 days.

Different letters in each evaluation indicates difference between treatments after Tukeýs test (p ≤ 0.05).

The inoculation of tomato increased shoot length in the range of 22.7–42.1% (for FCCT 199-2 and FCCT 16 treatments, respectively) and no differences among treated plants were detected (Table 3). All inoculated plants showed an increase in the fresh shoot weight (higher than 220% in comparison with the control), and FCCT 16 inoculated plants showed the highest growth. A similar trend was found for the dry weight of the shoot, with a range of 53.91–135.6% (Table 3).

The inoculation of tomato plants with FCCT 16 and FCCT 199-2 strains increased fresh root weight significantly and FCCT 16 inoculated plants showed the highest percentage in root growth. It is further noted that all the inoculated plants showed greater proliferation of lateral roots in comparison with the control plants (Fig. 2).

3.5.2

3.5.2 Effect of Trichoderma on plants inoculated with F. oxysporum

After 15 days of inoculation with the pathogen, the oldest leaves of the control tomato plants showed the typical symptoms of chlorosis in the main and secondary petioles, as well as the progression of propagation throughout the plant. In contrast, plants obtained from seeds coated with each of the Trichoderma strains, showed a reduction of leaf disease between 10 and 30% (Fig. 3), although tomato plants inoculated with FCCT 363–2 displayed the best performance.

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Fig. 3

Disease evaluation as percentage of healthy leaves on tomato plants treated with strains FCCT 16, FCCT 58, FCCT 199-2, FCCT 363-2 after 15 days of inoculation with Fusarium oxysporum. Mean averages (n = 3). Different letters on bars indicates statistical differences between treatments after Tuckeýs Test (P ≤ 0.05).

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4 Discussion

The Trichoderma genus comprises a large number of soil borne filamentous fungi that are widely used as inoculants for their health benefits to plants, such as conferring better growth, disease resistance or tolerance to abiotic stress for their hosts.

In Argentina, as in the rest of the world, there is a growing interest in developing commercial products based on Trichoderma to be used as amendment and biocontrol agents. However, the study of native strains is still poorly investigated, so the selection of strains could be carefully targeted to improve their potential under field conditions. The beneficial effects of bioinoculants could also depend on the ability of preserving indigenous fungi from invasive microorganisms, in order to keep the native microbial composition finding new strains adapted to local agroecological conditions.

Although the use of chemical pesticides is common in the management of tomato crops and may alter the recovery of fungi from the soil, some Trichoderma species are more tolerant to chemicals than others (Nowara and Radwan, 2017). In this study T. brevicompactum, T. gamsii and T. harzianum were isolated from agricultural soils, usually treated with fungicides, and also from pristine soils. All strains were able to sporulate, and abundant chlamydospores were found which have a potential role as inoculum (Chet, 1987). Nineteen T. harzianum strains were identified and all of them showed inhibition of F. oxysporum growth between 20 and 100%, but four of them FCCT 16, FCCT 42, FCCT 58 and FCCT 363-2, were highlighted because showed values up to 80% (Fig. 1). In agreement with other studies, T. harzianum is reported to be the most aggressive specie against pathogens, suggesting its high potential as an effective agent of biocontrol (Siddiquee et al., 2009).

The production of plant regulators by microorganisms is an important mechanism often associated with growth stimulation, since it is probably a way in which Trichoderma improves the development of plants. The molecular basis that underlies this subject is still unclear. In this sense, our results are being in agreement with studies where Trichoderma species were reported as producers of IAA being part of their metabolism (Contreras-Cornejo et al., 2009, 2011). We determined that all Trichoderma strains produced IAA (data not shown) and interestingly, four of them showed to be the highest IAA producers (between 13.24 μg/ml and 24.3 μg/ml) (Table 2). The biosynthetic pathways of this phytohormone in microbes and plants are highly similar and either tryptophan (Trp)-dependent or Trp-independent pathways were detected. The production of IAA is in general increased several times by the addition of tryptophan or its derivatives in the culture medium. From our results, we highlighted that even without the addition of indole derivative compounds, we measured quantities of IAA as high as reported values, when 200 µg/ml of tryptophan was amended (Gravel et al., 2007). Moreover, it is remarkable that the strains that were higher producers of the phytohormone were also good biocontrollers of F. oxysporum, as confirmed in both in vitro and in vivo assays (Figs. 1 and 3).

Phosphorus is an essential nutrient required for plants, and its bioavailability is associated with increases in plant growth (Richardson, 2001). In our study, the in vitro disappearance of tri-calcium phosphate could indicate a good potential of the strains to solubilize the inorganic bound phosphate. The four selected strains as higher IAA producers (FCCT 16, FCCT 58, FCCT 199-2 and FCCT 363-2), showed this ability in liquid culture reaching 215.8–288 µg/µl of soluble phosphorus (Table 2); comparable to that of other studies carried out by Kapri and Tewari (2010). We demonstrate the sequestration of tri-calcium phosphate by Trichoderma and hypothesize that an available form of the nutrient could be present in close proximity to the roots. Probably, this ability to make available insoluble nutrients could have been supported by the production of different organic acids and phosphatase enzymes secreted by the fungus (Gravel et al., 2007). This indicates an additional key property to select more efficient strains. We found that the four Trichoderma strains that effectively produced IAA and were able to solubilize inorganic phosphorus also contributed to improve tomato plant growth after inoculation (Table 3). In plant assays, each strain increased the potential capacity for photosynthesis, since the inoculated plants showed SPAD readings of approximately 10.2–52.4% and the leaf area between 1.4 and 2.3 times over the control. These data could indicate a more efficient photosynthesis. All the inoculated plants showed growth promotion on shoots and roots and, surprisingly, the most outstanding effect was found in the dry weight of the roots (between 133 and 400%). We found changes in root development as a modification of root architecture, probably increasing the area suitable for the microbial colonization that extends the root system for nutrient uptake (Berg, 2009; Contreras-Cornejo et al., 2009). As previously reported, Trichoderma interferes with plant development, disturbing their auxin balance. So, based on our results, we could suggest that there is a link between IAA secreted by the fungus and growth of tomato plants.

Trichoderma interacts with other rhizosphere microbes influencing the protection against diseases, plant growth and yield. It is reported that some species are able to produce several plant defenses eliciting microbe associated molecular patterns (MAMPs) such as xylanases, swollenins, peptaibols and low molecular weight compounds (Druzinhina et al., 2011; Zeilinger et al., 2016). There is a special interest in those compounds with antibiotic activity, since they are more possibly effective in biological control. We found that the Trichoderma selected strains were able to reduce the F. oxysporum disease symptoms on tomato plants between 10 and 30% (Fig. 3). Probably, a priming effect could be exerted by the fungus associated with the root system. It is reported that in primed plants, defense responses are not directly activated, but are enhanced upon pathogens or insects, resulting in faster and stronger resistance reaction (Van Wees et al., 2008). Successfully, the benefits of priming have emerged as a promising strategy in disease management (Beckers and Conrath, 2007).

In summary, our results suggest that the Trichoderma strains from the Argentine Pampas stimulate the growth of tomato plants through the production of phytohormones, increasing the leaf area and probably photosynthesis and the uptake of phosphorus. As a result, a more vigorous plant could show a priming effect capable to favor the biocontrol against F. oxysporum. Although we consider that further experiments should be carried out to evaluate the effects of Trichoderma on adult plants and fruit yield, both the FCCT 16 strain and the FCCT 363-2 strain can be highly recommended for their properties. These can be properly formulated and applied in a sustainable tomato production.