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Introduction
Climate change and agricultural malpractices such as excessive use of fertilizers and pesticides have heightened the effects of abiotic stresses on crop productivity and ecosystem degradation (Begum et al., 2019). In this context, there is an urgent need for the application of environmentally-friendly management techniques such as the use of arbuscular mycorrhizal fungi (AMF) for enhancing crop productivity (Khaliq et al., 2022). These symbiotic fungi are some of the most commonly occurring living organisms in soil, producing benefits for the plant-soil ecosystem in terms of growth, development, stress tolerance, soil pollutant remediation, C-sequestration, food security and agricultural sustainability (Ortas & Rafique, 2018). Moreover, the symbiosis improves the supply of water and macronutrients, such as P and N, to the host plant with benefits for both partners (Azcón-Aguilar & Barea, 2015).
Arbuscular mycorrhizal fungi (AMF) interact with almost all organisms in the mycorrhizosphere, including saprophytic fungi; however, few studies have been carried out on their synergistic interaction, especially with those microorganisms belonging to the same P cycle. Saprophytic fungi live on the rhizoplane and mycorrhizosphere of plants and procure their nutritional requirements from organic matter and other elements in the soil (Azcón-Aguilar & Barea, 2015). Some of these fungi benefit plant growth by transforming fixed P into more available forms through mechanisms that include the release of chelant organic acids and phosphatase enzymes (Andrino et al., 2021; Rawat et al., 2021).
On the other hand, it has been observed that when microorganisms act together, they provide greater benefits to the plant than in isolation (Perea et al., 2019). In this context, Castillo et al. (2013) reported that inoculation of chili pepper with the native AMF Claroideoglomus claroideum accelerated fruit ripening along with increasing yield and quality, while co-inoculation with Penicillium albidum favored the development of a higher fruit number. Additionally, mycorrhizal inoculation alters the root architecture and some studies have shown that the nutrient absorption capacity of inoculated roots is enhanced in comparison with non-inoculated ones (Ortas & Rafique, 2018).
Therefore, the objective of this work was to evaluate the potential of two saprophytic phosphofungi to promote the development of native potato minitubers when inoculated with Claroideoglomus claroideum.
Materials and methods
Experimental design
Four mycorrhizal treatments were used for the trial: 1) C. claroideum (T0), 2) C. claroideum + Talaromyces pinophilus (T1), 3) C. claroideum + P. albidum (T2), and 4) C. claroideum + T. pinophilus + P. albidum (T3). Each of the four treatments had five replicates resulting in a total of 20 experimental units.
Biological materials
The C. claroideum inoculum used was a native morphotype isolated from agricultural acidic soils in Southern Chile (Castillo et al., 2009). The AMF inoculum consisted of a mixture of rhizospheric soil containing spores, hyphae and fragments of colonized roots (with an average potential of 50 spores∙mL-1).
Two saprophytic phosphofungi were used (as a suspension of spores): P. albidum (2.7 x 107 CFU in 750 mL) and T. pinophilus (8 x 107 CFU in 750 mL), belonging to the fungal collection of the Department of Chemical Sciences and Natural Resources of the Universidad de la Frontera, Chile (Morales et al., 2011).
The soil used was an acidic Andisol obtained from the Unidad de Docencia Práctica Pillanlelbún located 15 km north of Temuco (38° 39’ S and 72° 27’ W). It had the following chemical characteristics: P Olsen 13 mg∙kg-1, K available 192 mg∙kg-1, pH 5.28, soil organic matter (SOM) 18.7 %, exchangeable Ca 4.04 cmol(+)∙kg-1, exchangeable Mg 1.06 cmol(+)∙kg-1, exchangeable Al 0.074 cmol(+)∙kg-1, and Al saturation 1.28 %. This soil was sieved at 2 mm, then mixed with quartz sand (70:30, v:v) and sterilized by tyndallization for 1 h for 3 consecutive days.
Potato explant multiplication
Prior to the establishment of the assay, a multiplication of explants of potato variety ‘Güicoña’ nodal segments was carried out in Murashige and Skoog (1962) solid medium. The aseptically sealed medium remained in the growth chamber for 30 d, with a photoperiod of 16 h of light and 8 h of darkness, 200 to 300 µE∙m-2∙s-1 of illumination and a temperature of 22 °C. For the acclimatization process, replicates of homogeneous plantlets were transferred to seedling trays with sterile peat located in containers covered with transparent plastic where they remained in the growth chamber for 15 d with moisture maintained through manual irrigation. Subsequently, the containers were moved to the greenhouse and the lid was replaced with plastic wrap.
Experimental setup
Potato plantlets in seedling trays with peat attached to the roots were transferred to 750 mL containers containing the sterile substrate inoculated with 10 mL∙of C. claroideum. For the inoculation with the saprophytic phosphofungi, two 3-cm holes were made in the substrate, on which 6.4 mL of suspension of the respective fungus was added. Every 15 d, 20 mL of a 0.055 M KNO3 solution was added to each jar. The harvest was carried out 108 days after establishment during the phenological stage of tuber development. During the assay, moisture was maintained at field capacity.
Variables evaluated
In the plant, the following characteristics were determined: height, shoot dry weight (SDW), root dry weight (RDW), minituber (mT) dry weight (mTDW), mT number, and P in the mT. Also, with a WinRHIZO® image analyzer, the distribution of root length according to diameter was determined.
In the soil, the following parameters were determined: labile-P (it was carried out with 0.5 M NaHCO3 pH 8.5 according to the methodology of Sadzawka et al. [2006], but mineralizing the extract to discriminate Pi and Po [Secretaría de Economía, 2001]), acid phosphatase activity (P-ase) (by hydrolysis of p-nitrophenyl-phosphate [Tabatabai & Bremmer, 1969] with modifications reported by Rubio et al. [1990] for volcanic soils), and total microbiological activity (it was determined by the fluorescein diacetate hydrolysis [FDA] method according to Schnürer and Roswall [1982]).
Results and discussion
The co-inoculation of the Andisol with C. claroideum and the two saprophytic phosphofungi (T3) significantly (P < 0.05) increased the height and SDW of the potato plants, by 67 and 110 %, respectively, in relation to T0, which was the treatment inoculated only with AMF. With soil co-inoculation of C. claroideum and only one saprophytic phosphofungus (T1 or T2), no significant differences (P > 0.05) were observed among treatments in plant height and SDW, although better results were observed with P. albidum. In the case of the mTDW, it is important to highlight the great difference obtained between T0 and T3, the latter increasing the weight by 211 %, with respect to T0 (Table 1).
Table 1 Potato parameters in an assay of inoculation with Claroideoglomus claroideum (T0), Claroideoglomus claroideum + Talaromyces pinophilus (T1), Claroideoglomus claroideum + Penicillium albidum (T2), and Claroideoglomus claroideum + Talaromyces pinophilus + Penicillium albidum (T3) cultivated in an Andisol.
| Treatment | Height (cm) | SDW (g) | RDW (g) | mTDW (g) | mT (N°) | P mT (mg P) |
|---|---|---|---|---|---|---|
| T0 | 32.5 bz | 0.71 b | 0.023 a | 1.18 b | 3 a | 2.22 b |
| T1 | 38.3 ab | 1.25 ab | 0.036 a | 2.50 ab | 3 a | 5.47 a |
| T2 | 49.7 ab | 1.38 ab | 0.032 a | 2.26 ab | 3 a | 4.27 ab |
| T3 | 54.3 a | 1.49 a | 0.035 a | 3.67 a | 3 a | 6.77 a |
| LSD | 13.26 | 0.68 | 0.02 | 1.17 | 1.22 | 1.95 |
SDW = shoot dry weight; RDW = root dry weight; mTDW = minituber dry weight; mT = minituber number; LSD = least significant difference. zMeans for each response followed by the same letter do not differ significantly (Tukey, P ≤ 0.05).
On the other hand, in potato plant root length, it was again observed that the greatest length was obtained with T3, without significant differences (P > 0.05) in relation to individual inoculation with the saprophytic phosphofungi (T1 or T2), but presented differences with T0. The T3 treatment, corresponding to the co-inoculation of C. claroideum with the two saprophytic phosphofungi, significantly increased the length of fine roots (diameter 0 - 0.25 mm) (Table 2). In general, it was observed in the root architecture that when the diameter increases, the length of the root decreases, but without significant differences (P > 0.05) among treatments (Table 2).
Table 2 Distribution of root length in an assay of potato inoculated with Claroideoglomus claroideum (T0), Claroideoglomus claroideum + Talaromyces pinophilus (T1), Claroideoglomus claroideum + Penicillium albidum (T2), and Claroideoglomus claroideum + Talaromyces pinophilus + Penicillium albidum (T3).
| Treatment | Root length (cm) | Diameter (mm) | |||
|---|---|---|---|---|---|
| 0 - 0.25 | 0.25 - 0.50 | 0.50 - 0.75 | 0.75 - 1.00 | ||
| T0 | 202 bz | 113 b | 56 a | 10 a | 6 a |
| T1 | 374 a | 233 ab | 96 a | 25 a | 8 a |
| T2 | 346 ab | 220 ab | 95 a | 20 a | 6 a |
| T3 | 385 a | 246 a | 96 a | 25 a | 7 a |
| LSD | 218.46 | 154.24 | 39.76 | 14.50 | 5.33 |
LSD = least significant difference. zMeans for each response followed by the same letter do not differ significantly (Tukey, P ≤ 0.05).
Among all interactions in a soil environment, fluorescein diacetate hydrolysis enzyme activity is commonly used as an early indicator of biological activity in soils. This hydrolysis is mostly accepted as an accurate, simple and fast method for measuring total microbial activity in a range of environmental samples, including soils (Patle et al., 2018).
Co-inoculation of C. claroideum with the two saprophytic phosphofungi (T3) significantly increased (P < 0.05) the microbial activity measured by FDA reflexing the highest microbial charge; however, simultaneously there was a decrease in the phosphatase activity of the soil (Table 3). On the other hand, the P availability from the different labile fractions of the soil was different depending on the inoculation treatment used in the trial. Co-inoculation of AMF with P. albidum (T2) significantly increased (P < 0.05) the total and inorganic labile-P in the soil, while, with the mycorrhizal fungus inoculated with T. pinophilus (T1), a drastic decrease (P > 0.05) in the availability of the nutrient was observed (Table 3).
Table 3 Microbiological activity and labile-P in an assay of potato cultivated in an Andisol, and inoculated with Claroideoglomus claroideum (T0), Claroideoglomus claroideum + Talaromyces pinophilus (T1), Claroideoglomus claroideum + Penicillium albidum (T2), and Claroideoglomus claroideum + Talaromyces pinophilus + Penicillium albidum (T3).
| Treatment | Microbiological activity | Labile-P (mg∙kg-1) | ||||
|---|---|---|---|---|---|---|
| FDA (µg∙g-1) | P-ase (µmol∙g-1∙h-1) | Total-P | Inorganic-P | Organic-P | ||
| T0 | 31.2 bz | 0.90 b | 12.9 b | 11.0 c | 1.91 a | |
| T1 | 28.5 b | 1.48 a | 10.5 c | 10.3 c | 0.2 b | |
| T2 | 34.6 b | 0.87 b | 18.4 a | 17.0 a | 1.4 a | |
| T3 | 43.7 a | 0.44 c | 15.7 a | 14.7 b | 1.0 a | |
| LSD | 5.92 | 0.27 | 1.80 | 1.43 | 0.32 | |
FDA = fluorescein diacetate; P-ase = acid phosphatase activity; LSD = least significant difference. zMeans for each response followed by the same letter do not differ significantly (Tukey, P ≤ 0.05).
The plantlets used for the assay were obtained by micropropagation of nodal explants of pathogen-free potato segments, with more fragile plants in the early stages of phenological development. The native ‘Güicoña’ variety was used in the study; however, in the country there is a great variety of different shapes and colors. Solano et al. (2013) reported that higher allelic richness in a variety must be conserved to promote biodiversity and also as a potential source of future potato varieties. In this regard, studies of this type can make an interesting contribution to local potato tuber seed growers working under sustainable conditions.
The greater growth of fine roots in the potato plants inoculated with C. claroideum together with the two saprophytic phosphofungi could suggest that these fungi acted as inducers of root development and thus allowed the plant to explore a greater volume of soil for the acquisition of nutrients. In this context, it has been reported that in soils with low P availability, plants can modify root architecture by increasing root hair development or lateral root growth (Niu et al., 2013). The formation of fine roots is critical for the uptake of nutrients and water from the soil; therefore, these fungi modified the root architecture to meet the water and nutritional requirements of potato plantlets. Sharma & Kaur, 2017; Toscano-Verduzco et al., 2020 reported the existence of microorganisms capable of producing plant growth hormones that play an important role in plant development, resulting in a plant-microorganism interaction which is a determining factor for the production of this type of hormone (Sun et al., 2019).
On the other hand, the decrease in P-ase observed in treatments T2 and T3 could be a result of the greater generation in the finest roots, since they allow a higher P absorption (PmT in Table 1), contrasting with the enzyme level which decreased in T2 and T3. It is known that P-ase exudation is inhibited with a high shoot/root P concentration and also that plants have evolved numerous morphological and physiological adaptations to cope with P limitation. These adaptations increase the roots’ P-uptake surface area, mobilizing unavailable soil P (Vance et al., 2003). However, some plants, instead of relying on processes such as enzyme exudation, modify the morphology or architecture of their roots (Lyu et al., 2016; Shen et al., 2018).
Currently, there is a great variety of AMF ecotypes, with C. claroideum, a mycorrhizal fungus isolated from Andisols in Southern Chile, standing out among them. In terms of sustainable agriculture, the benefit that could be obtained with a joint inoculation of native potatoes with solubilizing fungi in highly fixing soils is noteworthy, possibly due to more effective P uptake. In this regard, the mobilization of P towards the mT in the saprophytic phosphofungi interaction with C. claroideum in this study shows that the interaction in the Andisol of C. claroideum with the two solubilizing phosphofungi (T. pinophilus and P. albidum) benefits the growth and yield of mT in potato plantlets. These results agree with what was reported by Sembiring et al. (2018) in oil palm seedlings, and Silitonga et al. (2018) in soybean and more concretely by Sembiring and Fauzi (2017), who reported in an Andisol that inoculation with T. pinophilus increased available P and potato production. In relation to inoculation with P. albidum, other studies confirm the observed benefits of cultivating clover, lettuce, and chili pepper in Andisols (Castillo et al., 2013). Consequently, inoculation with P. albidum would be applicable to various other types of plants, where T. pinophilus would have a possible solubilizing potential for P.
The quality of soil depends in part on its natural chemical and biological composition, and also on the changes caused by human use and management. Strategies based on biological indicators would be a suitable tool to evaluate the sustainability of the soil ecosystem. Soil enzyme activity studies are good and quick indicators to measure the ecosystem status and quality of soils (Patle et al., 2018). The phosphatase enzyme, produced by plants and microorganisms, plays an impotant role in the soluble organic P mineralization, being essential for P cycling in deficient soils.
Inoculation of C. claroideum with T. pinophilus increased P-ase levels in the soil, while the inoculation of the AMF with the two saprophytic phosphofungi significantly increased the FDA, which is indicative of a higher microbial biomass. The results are noteworthy since the FDA hydrolysis enzyme in the soil could serve as an indicator of the soil’s potential to support biochemical processes, which are essential for maintaining soil fertility as well as soil health (Patle et al., 2018). In this sense, the beneficial increase in microbial biomass could induce a concomitant increase in P, N and S in their cells, producing a transient inmobilization of these nutrients for further mineralization.
Finally, the indiscriminate use of chemical fertilizers and pesticides has caused considerable environmental damage in recent years which, together with the growing demand for food, requires the use of increasingly productive and efficient agricultural systems. Several studies have shown that the application of plant growth-promoting microorganisms can be a valid substitute for chemical industry products and represents a valid eco-friendly alternative for avoiding the excessive application of soluble phosphate fertilizers. However, due to the complexity of interactions created with the numerous biotic and abiotic factors, the different formulates often show variable effects (Hernández-Fernández et al., 2021) and consequently there is an urgent need to develop new technological tools focused on mobilizing P soil fractions. especially those with higher lability. Our results showed that the interaction between AMF and T. pinophilus can improve plant growth by 13 to 56 % and P uptake by 15 to 69 %.
Conclusions
It is concluded that co-inoculation of C. claroideum with the two saprophytic phosphofungi enhanced mass and P content of ‘Guicoña’ native potato minitubers, together with root length and thinness and soil enzymatic activities when growing in an Andisol.
The results of this study supply background information for sustainable agriculture which requires satisfying nutritional aspects of plants through the use of eco-friendly management techniques that reduce the use of synthetic fertilizers, in this case by applying native beneficial microorganisms to improve potato yield.
