Characterization of dark septate endophytic fungi Periconia macrospinosa isolated from roots of sugarcane in São Paulo, Brazil

Abstract

Dark Septate Endophytic (DSE) fungi can benefit plants by optimizing nutrient uptake, biosynthesis of phytohormones-like compounds, and stress relief such as toxic metals. The objective was to characterize in vitro 57 strains of the DSE Periconia macrospinosa isolated from sugarcane roots, indicating the most promising in solubilizing phosphate sources, growing in different metal and vinasse contents, and producing molecules related to indoleacetic acid (IAA). The strains were from the Embrapa Agrobiology Fungi Collection. Over 35% of the strains solubilize calcium phosphate, highlighting A356 and A155. No strain solubilizes aluminum phosphate. Three strains did not grow in the presence of Cd (A333, A334, A163), but A226, A332, and A423 stand out showing high growth with Cd. All strains grew in the presence of Cu, Zn, and Vinasse. A163, A164, and A328 were even stimulated by Cu addition. A331 and A335 showed a marked growth decrease with Zn. All strains are highly adapted to grow in vinasse presence. A257 and A226 produced the highest amount of IAA. The most biotechnological potential strains are indicated by presenting high growth under Cd, Cu, Zn, and vinasse stress, associated with a high tolerance index to these pollutants, further calcium phosphate solubilization, or IAA production.

Key words
auxin production; dark septate endophytes; heavy metals; phosphate solubilization; pleosporales; vinasse

INTRODUCTION

Endophytic fungi live internally in plant tissues, at least during a period of their life cycle. Among them, Dark Septate Endophytes (DSE) are an important group of asexual ascomycete fungi that frequently colonize plant roots and have characteristic melanized hyphae and microsclerotia (Mandyam & Jumpponen 2014, Akhtar et al. 2022). DSE are involved in several benefits for plant development, being able to promote plant mineral nutrition, mainly by solubilization of phosphate sources (Wang et al. 2015, El-Ghandour et al. 2018) and increasing phosphorus absorption (Zhu et al. 2018), producing similar substances to plant growth hormones (Hamayun et al. 2009, Azevedo 2014), increasing the plant tolerance to metals excess (Azevedo 2014). These capabilities make DSE useful for the phytoremediation of contaminated areas (Azevedo 2014) and for fertilizing plants in a more sustainable way.

Among the fungal species considered DSE, Periconia macrospinosa has received attention due to its frequent isolation from roots, commonly related to grasses, such as wheat, barley, and sugarcane (Mandyam & Jumpponen 2015, Raza et al. 2019, Fors et al. 2020). P. macrospinosa can use organic and inorganic sources of macronutrients and promote root and shoot growth (Mandyam et al. 2010). There are also reports of positive effects of its inoculation on tomato plant growth (Yakti et al. 2018) and on improving salinity tolerance, increasing chlorophyll concentration, and antioxidant enzymatic activity in Barley subjected to water and saline stresses (Moghaddam et al. 2021). In addition, P. macrospinosa also acts as a biological control agent (Høyer et al. 2022) and assists nutrition processes through the solubilization of phosphate in the soil (Faria et al. 2016).

Brazilian agriculture is a world leader in the use of bio-inputs (Fischer et al. 2023), and selected microorganisms used as bio-inputs promote plant development (Souza et al. 2015), reduce the environmental impacts of crops (O’Callaghan et al. 2022), improve soil quality and plant health (Alori et al. 2017), reduce dependence on pesticides (Silva et al. 2022) and fertilizers (Abd-Alla et al. 2023) and promote savings in production costs (Oliveira et al. 2022). The development of bio-inputs requires extensive studies in the field, but initial laboratory studies that select microorganisms with the potential for the development of bio-inputs are very important in the rapid development of these biotechnologies. Among these basic studies, the in vitro characterization of microorganism isolates with biotechnological potential allows a rapid selection of isolates according to the desired objective for the bio-input. Microorganisms with beneficial potential to promote plant growth, such as P. macrospinosa, can have strains selected rapidly in vitro for functions such as phosphate solubilization and IAA synthesis, which has significant implications for advancing research seeking the use of bio-inputs to improve the sustainability of Brazilian agriculture.

For the continuity and advancement of studies based on the isolation of a large number of strains of P. macrospinosa from sugarcane roots (Fors et al. 2020) in the State of São Paulo, Brazil, this study had the objective of characterizing in vitro 57 strains of dark septate endophytic fungi P. macrospinosa in terms of their capacity to solubilize calcium and aluminum phosphate, to grow in different contents of cadmium, zinc, copper and vinasse and to produce molecules related to indoleacetic acid (IAA).

MATERIALS AND METHODS

Biological material

Seven in vitro experiments were carried out with 55 to 57 strains of P. macrospinosa isolated from sugarcane roots in São Paulo State, Brazil (Table I). All strains, belonging to Johanna Döbereiner Biological Resources Center (CRB-JD) from Embrapa Agrobiology, were isolated and identified by Fors et al. (2020).

Table I
Strains of Periconia macrospinosa isolated from sugarcane roots (Saccharum spp.), were collected in different municipalities in São Paulo State, Brazil, with the respective climate (Köppen-Geiger) and collection site biome. All strains are registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) in code A86D7CF. Cfa = Humid subtropical climate. Cwa = Monsoon-influenced humid subtropical climate. Aw = Tropical wet and dry or savanna climate.

Periconia macrospinosa strains	Municipalities	Climate	Biome
A111	João Ramalho	Cfa	Mata Atlântica
A113, A115, A124, A125, and A126	Quatá	Cfa	Mata Atlântica
A154, A155, A156, A157, A158, A159, A160, A161, A162, A163, and A164	Macatuba	Cfa	Mata Atlântica
A189, A190, and A191	Lençóis Paulista	Cfa	Cerrado
A224, A225, and A226	Piracicaba	Cfa	Mata Atlântica
A235, A236, A237, and A238	Pirassununga	Cwa	Cerrado
A257, A258, A259, and A260	Santa Lúcia	Cfa	Cerrado
A302	Motuca	Aw	Cerrado
A314, A315, A316, A317, and A318	Ribeirão Preto	Aw	Cerrado
A327, A328, A329, A330, A331, A332, A333, A334, A335, A336, A337, A338, A355, and A356	Igarapava	Aw	Cerrado
A372 and A373	Severínia	Aw	Mata Atlântica
A423, A424, and A447	Valparaíso	Aw	Mata Atlântica
A499	Ipaussu	Cfa	Mata Atlântica

All strains of P. macrospinosa isolated by Fors et al. (2020) were initially cultured on malt extract-agar medium - MEA (Malt extract 20 g.L^-¹; Dextrose 20 g.L^-¹; Peptone 1 g.L^-¹; Agar 15 g.L^-¹ and pH adjusted to 6.0) from colonies preserved by the Castellani method at CRB-JD. The selection criterion for the 57 strains used in this study was to choose those that showed growth in this initial culture from Castellani. Subcultures incubated at 28 °C in the dark from these initial cultures were made before the experiments, where colony discs measuring 8 mm in diameter were cut and used as inoculants in the various experiments carried out to characterize the strains, which will be described further below.

All experiments were conducted using the strains as treatments. The treatments were arranged in a completely randomized design with six replications. The experimental unit was a Petri dish when the medium was solid or a Falcon tube when the medium was liquid. The data from each experiment were checked for normality using the Shapiro-Wilk W test and subjected to analysis of variance, grouping means (P≤0.05), and regression analysis using the Sisvar 5.6 program (Ferreira 2011). The cluster analysis method used for grouping means was the Scott-Knott test (Scott & Knott 1974) and the means group formed were called “SK-groups”.

Phosphate solubilization experiment

The phosphate solubilization experiment was carried out in Petri dishes using solid Pikovskaya agar (PVK) medium (yeast extract 0.5 g.L^-¹, dextrose 10 g.L^-¹, (NH₄)₂SO₄ 0.5 g.L¹, KCl 0.2 g.L^-¹, MgSO₄.7H₂O 0.1 g.L^-¹, MnSO₄.H₂O 0.0001 g.L^-¹, FeSO₄.7H₂O 0.0001 g.L^-¹, and agar 15 g.L^-¹) (Pikovskaya 1948). In the experiment with calcium phosphate, 5 g.L^-¹ of Ca₃(PO₄)₂ were added to the PVK medium and the pH was adjusted to 7.0. In the experiment with aluminum phosphate, 3.93 g.L^-¹ of AlPO₄ and 5.37 g.L^-¹ of CaCl₂ were added to the PVK medium, and the pH was adjusted to 5.0.

A positive control treatment with the A521 strain of Aspergillus niger was added to the phosphate solubilization experiments together with the treatments of P. macrospinosa strains. The strain A521 belongs to Johanna Döbereiner Biological Resources Center (CRB-JD) and was selected among 11 other A. niger strains from the CRB-JD for presenting the largest phosphate solubilization halo in the PVK-calcium phosphate medium. A. niger is a species known to solubilize phosphates due to its production of citric acid, (Barroso & Nahas 2013, Mendes et al. 2013, Chandraker et al. 2014, Cholke Snehal et al. 2019).

Colony disks from each strain were inoculated and Petri dishes were incubated at 28 °C for 72 hours. The colony and the solubilization halo (when present) diameters were taken by two transversally opposite measurements. These measurements were after 48 hours and 72 hours of growth with a digital caliper. The mean colony diameter and the mean solubilization halo diameter were calculated from the two opposite diameter measurements and were used to calculate the solubilization index (SI) of each isolate using the formula (1) (Edi-Premono et al. 1996).

$$SI=\frac{\text{mean halo diameter}+\text{mean colony diameter}}{\text{mean colony diameter}}$$ (1)

The calculated solubilization indexes (SI) were classified according to Ruiz Berraquero et al. (1976) where SI<2 cm is a low solubilization index; SI with values between 2 cm and 4 cm is a medium solubilization index and SI>4 cm is a high solubilization index.

Excess metal tolerance experiments

The metal tolerance experiments were carried out in Petri dishes with a solid MEA medium. Levels of a metal were added to the medium in each experiment as shown in Table II. Metal levels in the medium were established based on literature data on fungal tolerance limits to metals (Zhang et al. 2008) or in initial tests observing growth inhibition of some Periconia strains. The control treatment was composed of level 0 of metal application in the MEA medium. The second level was above the acceptable limit for soils or substrates, where there was already some inhibition of fungal growth. The third level was a level already reported in the literature to be toxic to fungi or that the colonies were highly inhibited.

Table II
Metal excess tolerance experiments with Periconia macrospinosa strains and the respective metal levels, reagent doses to medium, and time of experimenting.

Experiment	Metal levels	Reagent doses	Time of experimenting
	mg L -1 of MEA	g L -1 of MEA	Hours
Cu tolerance	0, 300, and 1000 of Cu	0, 1.178, and 3.929 of CuSO4.5H2O	120
Cd tolerance	0, 400, and 500 of Cd	0, 0.912, and 1.141 of 3CdSO4.8H2O	96
Zn tolerance	0, 400, and 600 of Zn	0, 1.742, and 2.628 of ZnSO4.7H2O	96

• MEA: malt extract-agar medium, Cu: copper, Cd: cadmium, Zn: zinc, Reagent doses: were applied from stock solutions during the medium dissolution, before autoclaving.

After the time of experimenting (Table II), colony growth was estimated by measuring their diameter by taking two transversely opposite measurements with a digital caliper. Using these two measurements, the mean colony diameter and the colony surface were estimated, considering the colony to be an ellipse. The colony growth speed was calculated, in mm per day, subtracting the initial diameter of the disk inoculated (8 mm) from the mean colony diameter, and then dividing the result by the time of experimenting.

The formula (2) of Oladipo et al. (2018) was used to calculate the metal tolerance index (TI), as follows:

$$TI=\frac{\text{mean colony diameter with metal}}{\text{mean diameter of the control colony}}$$ (2)

The calculated tolerance indexes (TI) were classified according to Oladipo et al. (2018) where TI between 0.00–0.39 is very low metal tolerance, TI between 0.40–0.59 is low metal tolerance, TI between 0.60–0.79 is moderate metal tolerance, TI between 0.80–0.99 is high metal tolerance, and TI ≥ 1.00 is very high metal tolerance.

Excess vinasse tolerance experiment

The vinasse used in the experiment contained only the fermented and distilled wort, without the addition of other residues. It had a pH of 4.08, electrical conductivity of 11.22 mS, total solids content of 9.35 g.L^-¹, and nitrogen content of 205.05 mg.L^-¹. The vinasse was incorporated during the preparation of the solid MEA medium. The reagents equivalent to one liter of MEA were diluted in water volume needed to complete vinasse volume to one liter in the proportions described in Table III and then autoclaved together.

Table III
Characterization of electrical conductivity and pH adjustment of medium treatments used in the excess vinasse tolerance experiment.

Treatments (proportion of vinasse per liter of MEA)	Conductivity (mS)	pH
MEA without vinasse (0%)	4.06	6.00
MEA + 400 mL L-1 of vinasse (40%)	4.25	6.00
MEA + 800 mL L-1 of vinasse (80%)	5.60	6.00

• MEA: malt extract-agar medium. The reagents equivalent to one liter of MEA were diluted in water volume needed to complete vinasse volume to one liter.

The fungal strains were tested in the three concentrations of vinasse (0, 400, and 800 mL.L^-¹), where the last two levels are equivalent in volume to the application of 800 m³ and 1600 m³ of vinasse per hectare. The concentrations were based on studies by Silva et al. (2014) and Costa et al. (2021) who found that even with extremely high applications of vinasse to the soil, sugarcane did not show a reduction in growth. The experiment was inoculated with the strains from 8 mm diameter discs and was incubated in the dark at 28 °C for 96 hours. The incubation time was defined for sufficient contrast between the vinasse dosages and before the mycelium growth covered the entire Petri dish. After incubation, two measurements of the colony diameter were taken, and the growth speed, in mm per day, and tolerance index (TI) were calculated, as described in the excess metal experiments. The TI is relevant for applying the tolerance classification of Oladipo et al. (2018) for strains regarding vinasse excess.

Molecules related to the indoleacetic acid (IAA) experiment

The experiment to characterize the fungal strains regarding the production of molecules related to the IAA growth regulator was conducted in Falcon tubes containing 20 mL of liquid MEA medium (Malt Extract Broth) supplemented with 100 mg.L^-1 of L-tryptophan (Rodrigues et al. 2007, Oliveira et al. 2012). The strains were inoculated from an 8 mm diameter disc taken from the margin of growing colonies in a solid MEA. Control tubes containing only the uninoculated liquid malt extract broth were incubated together with the others. The tubes were incubated in the dark at 27 °C, 160 rpm for 120 h. The analysis of molecules related to the IAA content was carried out in supernatant aliquots after medium centrifugation (5000 rpm for 5 minutes), according to a modification of Sarwar & Kremer (1995) methodology described hereafter. Three supernatant aliquots of 150 µL were taken (triplicate) from each Falcon tube. To each aliquot was added 100 µL of Salkowski’s reagent (5 mL of HClO₄ 70%+ 100 µL of FeCl₃ 0.5 M) and left to react in the dark for 30 min. The color development was read on a plate spectrophotometer with absorbance at 536 nm (Rodrigues et al. 2007). The fresh mycelium mass and the molecules related to the IAA produced per mycelium mass unit were quantified.

RESULTS

Calcium phosphate solubilization experiment

This study evaluated 55 strains of P. macrospinosa (Appendix - Table AI). All strains showed mycelial growth in PVK medium supplemented with calcium phosphate, pH 7.0. The strain A521 (Aspergillus niger), used as a positive control for solubilization, showed greater growth in mean colony diameter (MCD) than all P. macrospinosa strains. Excluding this control, the Scott-Knott test (P≤0.05) separated the P. macrospinosa strains into 5 to 6 growth SK-groups, which are marked by different letters in Appendix - Table AI.

The strains A333 and A257 showed higher growth than the other strains after 48 hours of incubation. These strains were followed by A191, A226, A236, A335, A337, and A499 as second growth SK-group. The SK-group with the lowest growth was the strains A125, A126, A155, A162, A235, A314, A316, A327, A330, A332, A338, and A356. After 72 hours, strains A161 and A333 showed the greatest growth, followed by a second SK-group of strains A190, A226, A236, and A258. The lowest growth SK-group has the strains A162, A235, A327, and A330. The daily growth rate in the first 48 hours (Figure 1) reflects the MCD result (Appendix - Table AI), as the initial colony size was the same for all treatments. However, after 72 hours, the strains showed variation in daily growth rate, showing a growth rate 2.5 times faster (Figure 1).

Figure 1
Daily growth rate of colonies of different Periconia macrospinosa strains during incubation between 0-48 and 48-72 hours on Pikovskaya agar (PVK) medium with calcium phosphate. Means followed by the same letter over the same color bars do not differ by the Scott-Knott 5% test. A. niger: Aspergillus niger strain A521 as a positive control of solubilization.

Eighteen P. macrospinosa strains showed a translucent halo of solubilization after incubation for 48 hours. After 72 hours, there were halos in 20 strains (A356, A327, A155, A235, A162, A316, A336, A423, A260, A157, A330, A158, A257, A238, A317, A154, A337, A224, A156, and A258). The positive control A. niger presented the largest halo, superior to all P. macrospinosa strains (Appendix - Table AI).

The solubilization index (SI) was higher in strains A155 and A356 than control A. niger, after 48 hours of incubation (Appendix - Table AI). Although A. niger had higher SI than the majority of P. macrospinosa strains except strains A235, A316, and A327. After 72 hours, the A356 strain remained with SI higher than the A. niger control. Strains with SI values equal to 1.00 did not solubilize calcium phosphate in the PVK medium. Among the 20 solubilizing strains of P. macrospinosa, there was a negative correlation between colony diameter and SI (r = -0.88) and a positive correlation between halo diameter and SI (r = 0.86).

Aluminum phosphate solubilization experiment

This experiment evaluated 57 strains of P. macrospinosa and all strains showed mycelial growth in PVK medium plus AlPO₄ and CaCl₂, pH 5.0. However, only A. niger (A521) presented a solubilization halo with a mean diameter of 18.78 mm and a solubilization index (SI) equal to 2.00. After incubation for 72 hours, A163, A335, A334, A333, A. niger, A315, and A332 had the largest mean colony diameter (18.0 to 20.3 mm), showing a higher daily growth rate (2.88 to 4.56 mm.day^-1). The A260, A159, and A161 P. macrospinosa strains are the second major growth SK-group in the PVK medium plus AlPO₄ and CaCl₂.

Excess metal and vinasse tolerance experiments

The tolerance experiments (cadmium, copper, zinc, and vinasse) evaluated 57 strains of P. macrospinosa.

Excess cadmium tolerance experiment

Strains A331 and A335 presented a larger mean colony diameter (A331) and colony surface (A331 and A335) than the other strains of P. macrospinosa in level 0, the control treatment without cadmium (Appendix - Table AII). When cadmium was present (Levels 400 and 500 mg.L¹), these two strains showed low growth, being in the SK-group marked with the letter ‘g’ in the Scott-Knott test (P≤0.05). The strain A226 had the largest diameter and surface of colony compared to the others in level 400 mg.L^-1. It was followed by a second SK-group composed of strains A423 and A332 (Mean colony diameter) added with strains A158 and A160 (Colony surface). The lowest growth SK-group was composed of strains A331, A335, and A329. At maximum level (500 mg.L^-1), the strains A226, A332, A157, and A423 had the highest growth. The second growth SK-group was composed of A328, A158, A161, and A160. The SK-group with the lowest growth was composed of A335, A329, and A331 like the level 400 mg.L^-1.

The strain tolerance indexes (TI) to cadmium are shown in Figure 2. The strains with the highest cadmium TI, based on the level 500 mg.L^-1, were A333, A334, A332, A336, A423, A163, A355, A226, A157, A161, A162, A337, A158, A126, A338, A328, A159, A160, A235, A225, A447, and A224. All these strains are classified as having “moderate tolerance”, according to Oladipo et al. (2018). The strains A154, A155, A329, A335, and A331 were classified as having “very low tolerance”. The A155 strain showed the greatest variation between TI between levels 400 and 500 mg.L^-1, where in lowest level showed “moderate tolerance” and in highest level “very low tolerance”.

Figure 2
Tolerance index (TI) to the pollutants cadmium, copper, zinc, and vinasse of different strains of Periconia macrospinosa at two pollutant concentrations in malt extract-agar (MEA) medium. Blue bars: lower concentration. Red bars: higher concentration. The same color bars with the same letters indicate that means do not differ by the Scott-Knott 5% test. Green lines indicate the limits of tolerance index classification according to Oladipo et al. (2018), 0.00 – 0.39: Very low tolerance, 0.40 – 0.59: Low tolerance, 0.60 – 0.79: Moderate tolerance, 0.80 – 0.99: High tolerance, and 1.00 – ≥1.00: Very high tolerance. Means in descending order of TI at the highest concentration (red bars).

Excess copper tolerance experiment

Strains A158, A237, A334, A355, A317, A318, A258, and A257 showed larger colony diameters and surfaces than other strains in MEA without added copper (Level 0) (Appendix - Table AIII). The slowest-growing strain was A163 with colonies of just 20 mm in mean diameter after 120 hours of incubation. When added 300 mg.L^-1 of copper, strains A162, A334, and A191 had the largest colony diameter and surface. With 1000 mg.L^-1 of copper, these three strains added strains A160, A316, A158, A330, and A499 formed the highest growth SK-group. The strains A113 and A331 consistently showed lower growth with copper in both levels, forming the lowest growth SK-groups.

The strain tolerance indexes (TI) to copper are shown in Figure 2. The strains A164 and A328 had the highest TI at level 300 mg.L^-1, while at 1000 mg.L^-1 were strains A163 and A164. All strains studied were classified as having “very high” or “high” tolerance to copper, according to Oladipo et al. (2018).

Excess zinc tolerance experiment

The A331 strain presented the largest colony diameter and, together with the A335 strain, the largest colony surfaces than other strains in the control medium without zinc (Appendix - Table AIV). Strains A333, A334, and A163 showed less growth in colony diameter and surface than other strains without zinc. At level 400 mg.L^-1 of zinc, the strains A318, A226, A158, A159, A373, and A156 were in the first SK-group with the highest colony diameter growth. For colony surface, the first SK-group was restricted to strains A318 and A226. The strains A155 and A331 were in the last SK-group with the smallest colony diameter. At level 600 mg.L^-1 of zinc, the strains A158, A302, A314, A315, A330, A161, and A373 had the largest colony diameters and surfaces and the Scott-Knott test separated the strains into a few SK-groups.

The strain tolerance indexes (TI) to zinc are presented in Figure 2. At level 400 mg.L^-1 of zinc, most strains were classified as having “high” or “very high” tolerance to zinc (Oladipo et al. 2018). The strains A423, A335, A329, A155, and A331 were classified as having “very low” tolerance to the level 400 mg.L^-1 of Zinc. At level 600 mg.L^-1, strain A163 showed the highest TI, followed by strains A158, A161, A162, A373, A372, A315, A314, A330, and A302, all these showing “high” tolerance. Most strains showed “very low” TI at level 600 mg.L^-1.

Excess vinasse tolerance experiment

Strains A257, A334, A331, and A155 showed greater colony diameter and surface than other strains in the control treatment without vinasse (Appendix - Table AV), while the strains. A314, A338, A317, and A316 showed the lowest growth in colony diameter and surface. Strain A334 stood out from the others, presenting a larger colony diameter and surface at levels 400 and 800 mL.L^-1 of vinasse. Also noteworthy are the strains A331, A257, and A335 that appear in SK-groups that succeed the strain A334 in one of the two variables. Strains A113, A338, A235, A163, and A190 are consistently in SK-groups with smaller diameters and surfaces of colony in both levels of vinasse.

The strain tolerance indexes (TI) to vinasse are presented in Figure 2. The strain A316 had the highest TI at level 400 mL.L^-1, followed by the A126 strain, both more tolerant to vinasse than the other strains. However, at level 400 mL.L^-1, all strains showed a “high” or “very high” TI according to the classification by Oladipo et al. (2018). Strains A316, A158, A317, A314, A373, A335, A334, and A125 were more tolerant than the other strains at level 800 mL.L^-1. The strains A115, A372, A355, A257, A190, A258, A155, A260, A111, A164, and A163 had the lowest TI at level 800 mL.L^-1 but still classified as “moderate” tolerance (Oladipo et al. 2018).

Strain mean daily growth rate from tolerance experiments at three different stress levels

The means of strains daily growth rate from all tolerance experiments (Cadmium, Copper, Zinc, and Vinasse) were analyzed in Figure 3. The strains cultivated in MEA medium without adding the analyzed stress factor resulted in a daily growth rate varying between 4.1 and 9.2 mm.day^-1. Strain A331 was the fastest growing, followed by strains A257, A237, A318, A226, A335, A157, A332, A154, A160, A191, A156, and A258 with a growth rate above 7.9 mm.day^-1. Strains A333, A356, A125, A126, and A163 were the slowest with a growth rate below 6.0 mm.day^-1.

Figure 3
Mean daily growth rate of different strains of Periconia macrospinosa colonies during incubation on malt extract agar (MEA) medium in the experiments with three levels of Cadmium, Copper, Zinc and Vinasse as stress factor. Means followed by the same letter do not differ by the Scott-Knott 5% test.

The strains had a daily growth rate between 2.3 and 6.7 mm.day^-1 in the intermediate established stress condition (medium level in the experiments). While in the highest stress condition (highest level) it ranged from 2.5 to 5.6 mm.day^-1. Strains A226, A158, A373, and A162 had a higher daily growth rate in medium-stress conditions and still maintained an outstanding daily growth rate in the higher-stress conditions. Strains A338, A329, A335, A163, and A331 showed the lowest daily growth rate in the medium-stress conditions and also in the highest-stress conditions.

Molecules related to the indoleacetic acid (IAA) experiment

This experiment evaluated 57 strains of P. macrospinosa for the production of molecules related to IAA (Appendix - Table AVI), which will henceforth be referred to simply as IAA. Strains A157, A156, and A158 showed greater mycelial mass production than the other lineages, followed by a second SK-group of the strains A115, A125, A113, and A126. The strains A237, A238, A257, A259, A258, and A260 had the lowest mycelial mass production.

Strains A302 and A226 promoted the highest concentrations of IAA in the MEA medium (Appendix - Table AVI), followed by a second SK-group with strains A355, A225, A499, and A257. The SK-group that promoted the lowest concentrations of IAA was of the strains A327, A126, A156, A328, A224, A164, A258, A191, A157, A111, A115, and A125, which did not differ from IAA concentrations in the uninoculated control medium.

The strain A257 promoted the highest amount of IAA per gram of mycelium (Appendix - Table AVI). The strain A226 was in a second SK-group and strains A225, A260, A237, and A259 were in a third SK-group. The strains A335, A329, A315, A316, A154, A336, A318, A327, A317, A159, A155, A328, A113, A126, A111, A158, A156, A115, A125, and A157 produced the least IAA per gram of mycelium. The difference between the strain that produced the most (A257 = 638 µg g^-1) and the one that produced the least (A157 = 105 µg g^-1) is 6 times greater.

DISCUSSION

Solubilization of calcium and aluminum phosphates

Eighteen P. macrospinosa strains showed the ability to solubilize calcium phosphate very early, presenting a translucent halo after 48 hours of incubation. After 72 hours, there were halos in 20 strains (Appendix - Table AI), which represents 36% of P. macrospinosa strains with solubilization up to 72 hours. Trichoderma spp isolates did not show solubilization up to 72 hours in PVK medium, but after 168 hours of incubation, 53% of the isolates showed solubilization (Prasad et al. 2023). The result obtained in this 20 P. macrospinosa strains with solubilization in a short incubation time corroborates the results of Faria et al. (2016) that the P. macrospinosa isolate FUN502A, obtained from Anacardium othonianum, showed solubilization of calcium phosphate in GYP medium (glucose, yeast extract, and peptone) after 72 hours of incubation. The precocity of solubilization may be related to the rapid daily growth rate of the strains (Figure 1). P. macrospinosa strains with faster daily growth rates must also exhibit earlier nutrient solubilization and absorption. P. macrospinosa presents colonies with more than 100 mm in diameter in just three weeks of incubation, superior to Leptodontidium sp. and Cadophora sp. which are other DSE species (Gaber et al. 2020).

The control A. niger was the only strain that combined high colony growth and high solubilization index (SI) associated with a high diameter of the solubilization halo. Among the 20 solubilizing strains of P. macrospinosa, there was a negative correlation between colony diameter and SI and a positive between halo diameter and SI. The characterization of fungal strains regarding their ability to grow and solubilize calcium phosphate helps in the rapid selection of strains with the desired potential to promote plant nutrition. Strains with greater growth and calcium phosphate solubilizing capacity have greater potential to assist assimilation of poorly soluble phosphate sources or recovery of fixed phosphorus, as suggested by Mendes et al. (2014). The selection of strains with greater solubilizing capacity is a biotechnological approach that promotes the metabolic capacity of growth-promoting fungi, expanding their potential for application in agriculture and environmental remediation (Kour et al. 2021). In addition to the 5 strains mentioned above, future solubilization studies should also include the strains A356, A327, A155, A235, A162, and A316 that stood out in SI and halo diameter. Particularly the A356 strain, which presented a higher SI than the A. niger control. Studies on insoluble phosphates solubilization by DSE fungi are still scarce in the literature. Vergara et al. (2019) reported that none of their DSE fungal isolates produced a solubilization halo in the GL medium. On the other hand, fungal isolates from genera Rhizopus, Penicillium, and Aspergillus, belonging to other fungal groups, have been extensively studied as solubilizers of calcium phosphate (Silva Filho & Vidor 2000, Balogun et al. 2022).

The SI value of strain A356 (2.02) also stands out considering fungal strains analyzed in other studies, such as Mahamuni et al. (2012), that obtained SI ranging between 1.13 and 1.59 for fungal strains isolated from sugarcane and sugarbeet, and Alam et al. (2002) that ranged from 1.53 to 1.80 for fungal strains from corn rhizosphere. The variable capacity of strains to solubilize phosphate is a consequence of the variety and quantity of organic acids that are produced by different fungal isolates (Whitelaw et al. 1999, Dighton 2007, Berthelot et al. 2019), and this difference occurs within genera, species, and isolates of the same species (Narsian & Patel 2000, Vergara et al. 2019). Aspergillus (Li et al. 2016) are capable of solubilizing phosphates in the soil by acidification, chelation, and stimulation of ion exchange reactions. These were certainly also the mechanisms used by the P. macrospinosa strains in the present study. The PVK medium supplemented with calcium phosphate, pH 7.0, proved to be efficient for this initial selection of P. macrospinosa strains, as all strains showed growth in it, in addition to reports on its efficiency for this purpose, when compared with other culture media. such as AYG, NBRIP, and NBRIY. (Pradhan & Sukla 2005, Rawat & Tewari 2011, Wang et al. 2020).

The screening of P. macrospinosa strains for their ability to solubilize aluminum phosphate found that all strains could not form a solubilization halo in the PVK medium plus AlPO₄ and CaCl₂, adjusted to pH 5.0. However, all strains grew in this medium, including strains that in the first 48 hours showed higher growth than A. niger, the positive solubilization control. A. niger did not present a halo in the first 48 hours, but it did after 72 hours. Although the P. macrospinosa strains did not solubilize aluminum phosphate in solid media in the present study, in the study of Faria et al. (2016) their P. macrospinosa strain solubilized 0.6 mg.L^-¹ of aluminum phosphate in liquid medium. Souchie et al. (2005) also observed low solubilization of AlPO₄ and Araxá apatite in solid media, but increased solubilization capacity of these phosphate sources in liquid media.

The fact that 20 strains of P. macrospinosa solubilized calcium phosphate, but not aluminum phosphate, is common in studies of the ability of fungal strains to solubilize. The solubilization of aluminum phosphate is less frequently observed in culture media when compared to calcium phosphate (Silva Filho & Vidor 2000). These authors observed that the majority of 56 strains of Rhizopus, Aspergillus, Paecilomyces, and Penicillium formed a halo of solubilization with calcium phosphate, but only five strains formed a halo with aluminum phosphate and none were able to solubilize iron phosphate.

Tolerance to metals and vinasse

Among the 57 studied strains of P. macrospinosa, only three strains (A333, A334, and A163) did not grow in the presence of cadmium (Cd). The other strains grew in the presence of Cd, highlighting A226, A332, and A423 which had high growth, in both levels of Cd (400 and 500 mg.L^-1). The tolerance index (TI), which is defined as the ability of a microorganism to survive the toxicity of a given material (Zafar et al. 2007, Iram et al. 2013), varied for strains, and due to the high Cd levels, the more tolerant strains presented “moderate tolerant” TI classification (Oladipo et al. 2018). DSE are reported to be tolerant to the excess of Cd in culture in vitro (Berthelot et al. 2016), and symbiosis with DSE are ubiquitous and abundant in stressful environments including places with high levels of toxic metals, exhibiting greater colonization intensity with increasing metal pollution (Pongrac et al. 2009, Li et al. 2011).

Tolerance to metals by filamentous fungi has been associated with its isolation sites, the toxicity and the concentration in the medium of the tested metal, and the innate isolate competence (Oladipo et al. 2018). The strain site of origin may explain its tolerance, however, the present P. macrospinosa strains, are all isolated from sugarcane roots cultivated in agricultural soils from São Paulo State, Brazil. Sugarcane crops can tolerate Cd stress, accumulating Cd in the plant, therefore being a potential Cd phytoremediator (Sereno et al. 2007, Zeng et al. 2017). Sugarcane, being one of the most important energy crops, producing abundant biomass, can also help high Cd soil bioremediation, particularly when associated with Cd-tolerant microorganisms. The most Cd-tolerant P. macrospinosa strains have biotechnological potential to favor sugarcane in soil with Cd overload brought by fertilizers or pesticides, or by the application of vinasse mixed with industrial effluents, or even by a mineralogical soil characteristic (Gadd & Sayer 2000). Agricultural soils with high Cd levels can be linked to several factors, including the use of phosphate rocks (Gupta et al. 2018). The ore processing to produce phosphate fertilizers increases the toxic metals concentrations in the final product, consequently, the application of phosphate fertilizers increases soil metals concentration (Khater 2008). Among the nine strains that presented the highest TI at the highest Cd level (A333, A334, A332, A336, A423, A163, A355, A226, and A157), five were isolated from Igarapava municipality (Table I), suggesting a selective pressure in this location to adapt the strains to higher Cd levels.

The tolerance to high levels of Cd observed here in some strains of P. macrospinosa may be related to its melanized color. Fungal melanin is a complex macromolecular compound, whose composition includes phenolic and aromatic compounds, providing rigid support to the mycelial cell wall, which increases the tolerance of darker fungi to various stressors, such as toxic metals, drought and oxidative conditions (Eisenman & Casadevall 2011, Berthelot et al. 2017). Among the few studies on Cd tolerance by DSE, there is the study of the Cd-tolerant DSE Exophiala pisciphila H93 strain, isolated from the roots of Arundinella bengalensis, which grew in a mine smelter in Yunnan province, China. This strain has remarkable tolerance to Cd, accumulating 3.57-4.93% of Cd in the mycelium, in a medium with 400 mg.L^-1 of Cd (Zhang et al. 2008). Furthermore, DSE fungi inhabit most roots of healthy plants in smelting mines in China with high concentrations of Cd (Zhang et al. 2008). According to Su et al. (2021), DSE sequestes Cd in chlamydospores and vacuoles, which limits the circulation of Cd in cells, reducing toxicity. The chromotropic acid (1,8-dihydroxynaphthalene), present in DSE melanin, helps in Cd tolerance through its immobilization in hydroxyl groups (Potisek et al. 2021).

The strains that had great growth in Cd presence associated with high Cd tolerance index (TI) were A226, A423, A332, A355, A158, and A157 (Appendix - Table AII, Figure 2), indicating that even though they were isolated from common agricultural conditions in sugarcane farming, they have biotechnological potential to be used as symbionts in future studies of phytoremediation or soil or water depollution. The availability of selected biological materials for removing toxic metals is highly desired, as it is cheap biotechnology, with high efficiency and often specific to the type of metal (Muñoz et al. 2006, Iram et al. 2015).

All 57 studied strains of P. macrospinosa growth at both levels of copper. Some strains (A163, A164, and A328) had little growth in colony diameter and surface in the absence of copper (Appendix - Table AIII). These strains showed superior growth with copper, resulting in a tolerance index (TI) greater than 1.22 (Figure 2), being classified as having a very high tolerance to copper (Oladipo et al. 2018). On the other hand, some strains had great growth in the absence of copper (A317, A355, A237, and A258) and showed a marked growth decrease in the presence of 1000 mg.L^-1 of copper, resulting in them being among those with the lowest TI. Analyzing both levels of copper, the strains that combined great growth in the presence of copper and the highest tolerance index were A164, A162, A191, A423, A190, A160, A334, A163, A499, A257, A333, and A330 (Appendix - Table AIII and Figure 2). These strains are the most promising for future biotechnological studies and possibly have a greater aptitude for copper bioremediation, as the amount of mycelium produced will be greater, and melanized DSE mycelia show strong tolerance and absorption to metals (Su et al. 2021, Shadmani et al. 2021). Copper-tolerant DSE strains may be important for use in agricultural areas where overuse of copper fungicides may have polluted soils with excess copper (Mackie et al. 2015).

A variety of mechanisms linked to fungal mycelium mitigate the toxicity of copper and other metals. Copper can be immobilized in the fungal wall by melanin (Gadd & De Rome 1988) or by chitin itself (Zapotoczny et al. 2007) or even precipitated by exudates outside the hyphae, forming phosphate and oxalate salts outside the mycelium. (Crusberg 2004). However, little is still known about the physiology and metabolism of DSE. Berthelot et al. (2017) found that copper treatment promoted the synthesis of melanin by DSE fungi. This need for copper by DSE fungi may justify why all strains in the present study showed growth, even with high copper levels in the medium, and even why some strains grew more in the presence of copper than in its absence.

All 57 studied strains of P. macrospinosa growth at both levels of zinc, as seen for copper. However, there was no stimulus to fungal growth due to the presence of zinc, as occurred with copper, and the tolerance indexes (TI) did not exceed 1.03 (Figure 2). Like with copper, the strains with high growth in the absence of zinc (A331 and A335) showed a marked growth decrease in the presence of 400 and 600 mg.L^-1 of zinc, being among those with the lowest TI. High concentrations of zinc are toxic to fungi, delaying mycelial expansion and making hyphae more branched, swollen, and septate (Lanfranco et al. 2010, Ayad et al. 2018), but in adequate concentrations it is an essential nutrient, being easily absorbed and metabolized (Fomina et al. 2005). Thus, at the lowest zinc level (400 mg.L^-1), most strains showed a high tolerance index (TI) and only a small group was sensitive to zinc at this concentration, namely A423, A335, A329, A155, and A331.

The strains that combined high growth with a high tolerance index were A158, A373, A161, A314, A372, A315, A162, A330, A163, and A302 (Appendix - Table AIV and Figure 2), being biotechnologically promising and should be further evaluated in future studies of zinc bioremediation or depollution. The biotechnological potential of DSE to help plants in soils polluted with zinc was proven by Zhang et al. (2013), who showed that among 29 plant species growing in slag piles with high zinc concentrations (31265.9 ± 6875.05 mg.kg^-1), 28 were colonized by DSE hyphae, except the species Rorippa indica, and 24 of them were also colonized with microsclerotia. Zhu et al. (2018) demonstrated that under metal stress, the tomato seedlings inoculation with two strains of DSE Phialophora mustea decreased the roots and shoots zinc accumulation. The metal hyperaccumulator plant Noccaea caerulescens increased its zinc bioremediation potential by inoculating DSE strains, with the strain Leptodontidium sp. Pr30 increased plant growth without affecting Zn content and the strains Phialophora mustea Pr27 and Leptodontidium sp. Me07 helped the plant accumulate more Zn, without significantly modifying its biomass (Yung et al. 2021). Therefore, DSE strains had the potential to increase the phytoextraction of metals from the soil.

All 57 studied strains of P. macrospinosa showed great growth at both vinasse levels, at least tripling the initial diameter of the inoculated colony after 96 hours of incubation. The fact that all strains present a “high” to “very high” tolerance index at the lowest level of vinasse (40% of the culture medium) and that the majority of strains still maintain the same tolerance index at the highest level (80% of the culture medium), suggests that P. macrospinosa strains are highly adapted to this byproduct of the sugarcane alcohol industry. P. macrospinosa could be multiplied on a large scale in a medium that uses this agroindustry byproduct. Only strains A113, A338, A235, A163, and A190 consistently showed lower growth in both levels of vinasse, suggesting less adaptation to the MEA medium supplemented with this residue. The strains that combined high growth with a high tolerance index, analyzing both levels of vinasse, were A334, A316, A158, A373, A125, A126, A335, A328, A356, A336, A259, A160, A331, A332, A423, A237, A162, and A161 (Appendix - Table AV and Figure 2), have biotechnological potential and should be evaluated in future studies.

The high tolerance to vinasse of almost all P. macrospinosa strains suggests an adaptation or selection of these fungi in the field modulated by vinasse applications in sugarcane crops or, even, to the characteristics of the cellular content of sugarcane, such as the salts and nutrients concentration, which can be maintained in the vinasse. Studies on the management of vinasse application and its effects on the endophytic microbial community are scarce, therefore the present study is important, as the evaluation of the influence of vinasse levels on DSE fungal strains is unprecedented. Rhizospheric microorganisms are potential bioindicators for soil quality during waste application, due to their biochemical and metabolic activities and because they are sensitive to environmental changes, providing a rapid response to adverse factors (Martins & Campos 2011). Perhaps the presence of P. macrospinosa in crops could be correlated in future studies with the situation of soil pollution by vinasse, due to its great adaptation to high concentrations of this residue.

The analysis of the average of all tolerance experiments (cadmium, copper, zinc, and vinasse) confirmed that strain A331 shows the highest average growth in the MEA medium, without stressful factors. However, A331 is the most sensitive strain to stressful factors, being on average the strain that grows least both in the intermediate stress and in the highest stress condition induced by the levels. Microbial cells generally survive stress by decreasing growth or entering dormancy (Geisel et al. 2011), which is a disadvantage in their biotechnological use to bioremediate polluted soils, but not all strains have the same tolerance strategy. The prototypical inverse relationship between stress resistance and growth rate could be the behavior of most individuals within a stressed microbial population (Geisel et al. 2011), but not of all isolates from this population, as verified for Salmonella isolates by Guillén et al. (2022). This was also verified in the present study for P. macrospinosa strains. Strains such as A331, A335, and A258 show high growth without stressful factors, but greatly reduce their growth in the presence of these factors. Strains such as A226, A257, A191, A332, and A160 also have high growth without stressful factors but do not excessively reduce their growth in the presence of stress, showing themselves to be more promising for biotechnological use, such as bioremediation, as they escape the prototypical behavior described above.

Production of molecules related to the indoleacetic acid (IAA)

P. macrospinosa strains promoted concentrations of molecules related to IAA in the medium ranged from 3.28 to 8.29 µg.mL^-1, which was at least 10 times higher than the concentrations verified by Faria et al. (2016) for the P. macrospinosa Fun502A strain, which produced 0.3 µg.mL^-1. However, in the Faria et al. (2016) study, the strain was cultivated in a different medium to that of the present study (Potato-Dextrose), for less time (only 72 hours), and without L-tryptophan medium supplementation, which stimulates IAA synthesis by microbial strains (Idris et al. 2007), and also the methodology for determining IAA in the Faria et al. (2016) study was different. Lestari et al. (2021), using a methodology similar to that of the present study, obtained IAA production by fungal isolates from sugar production residues similar to those obtained here for P. macrospinosa.

The MEA medium L-tryptophan supplemented in the present study may have guaranteed the IAA evaluation success. IAA can be synthesized through two routes, one using L-tryptophan as the precursor (Rojas Contreras et al. 2010) and the other being independent of tryptophan (Kochar & Srivastava 2012). Thus, root exudates containing L-tryptophan stimulate the synthesis of IAA by microorganisms, which stimulates plant root growth, suggesting a close symbiotic relationship (Idris et al. 2007). Therefore, the production of IAA is an important functional trait of growth-promoting symbiosis, as it allows the plant to maximize root system development, favoring its nutrition (Goswami et al. 2014). Endophytic fungi play an important role in this context, as they are considered a source of natural bioactives, including IAA (Khan et al. 2010, Korejo et al. 2014).

DSE fungi are often reported as producing IAA. Hou et al. (2021) observed that under saline stress the DSE fungi Paraphoma chrysanthemicola and Bipolaris sorokiniana increased the IAA content in the roots of Artemisia ordosica. Other studies report several DSE species as producing IAA under stress conditions (Waqas et al. 2012, Priyadharsini & Muthukumar 2017, Qiang et al. 2019), suggesting IAA production from P. macrospinosa strains may be stimulated in stressful situations in sugarcane fields, such as water deficit.

The production of mycelial mass greatly affected the IAA production per gram of mycelium in the present study, verifying a negative correlation between these variables (r = -0.82). Thus, all strains in the highest mycelial mass group (A157, A156, A158, A115, A125, A113, and A126) were in the group with the lowest IAA production per gram of mycelium. This makes it difficult to select strains using only the IAA ratio per gram of mycelium so it was also chosen to observe strains that produced a high medium concentration of IAA associated with a greater production of mycelial mass, which would result in a good performance in promoting plant growth. These strains were A302, A355, A337, A124, A499, and A315 (Appendix - Table AVI). Therefore, these six strains should be added to the strains that produced greater amounts of IAA per gram of mycelium (A257, A226, A225, A260, A237, and A259). These twelve strains with biotechnological potential need to be evaluated in the future in the presence of plants, as in the study by Naureen et al. (2022), and the effect of its extracts, as in the study by Turbat et al. (2020).

Among the strains evaluated in the present study, strains A257 and A260 were the only ones that associated high IAA production with calcium phosphate solubilization. The main effect of IAA produced by rhizospheric microorganisms is the promotion of root growth. Which can have a direct effect on increasing phosphate absorption. Therefore, if these two growth promotion strategies (AIA production and phosphate solubilization) are associated, the benefit to the plant could be additive (Richardson 2001). Studies have indicated that strains with greater IAA production may be associated with a greater decrease in the amount of tricalcium phosphate in vitro (Souchie et al. 2007), and that strains that solubilize inorganic phosphate, and are good IAA producers, better promote the plant growth (Bader et al. 2020).

CONCLUSIONS

Fifty-seven strains of Periconia macrospinosa, isolated from sugarcane roots cultivated in São Paulo, Brazil, were characterized in terms of solubilization of calcium phosphate, tolerance to cadmium, copper, zinc, and vinasse, and production of molecules related to indoleacetic acid (IAA). The most promising strains for advanced biotechnological application studies are highlighted below. Strains A356, A327, A155, A235, A162, A260, A155, A336, A423, and A316 should be included in phosphate solubilization studies as they have a high capacity for calcium phosphate solubilization or prominent growth associated with solubilization. Strains A226, A423, A332, A355, A158, and A157 should be included in studies with cadmium. Strains A164, A162, A191, A423, A190, A160, A334, A163, A499, A257, A333, and A330 in study with copper. Strains A158, A373, A161, A314, A372, A315, A162, A330, A163, and A302 in zinc studies. Strains A334, A316, A158, A373, A125, A126, A335, A328, A356, A336, A259, A160, A331, A332, A423, A237, A162, and A161 in studies with vinasse. These strains combine great growth in the presence of the stressor and a higher tolerance index. Strains A302, A355, A337, A124, A499, A315, A257, A226, A225, A260, A237, and A259 should be included in advanced studies to promote plant growth as they produce a higher concentration of IAA-related molecules associated with high growth mycelial and high amount of IAA per gram of mycelium.

ACKNOWLEDGMENTS

The authors thank the technician Itamar Garcia Ignácio from Embrapa Agrobiologia for his assistance in conducting the experiments, the Programa de Pós-Graduação em Agronomia - Ciência do Solo, Universidade Federal Rural do Rio de Janeiro for the opportunity to develop this study as part of the first author dissertation, the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the first author scholarship, and the Embrapa project 10.20.02.009.00.00 - Conservation of Microorganism Collections at Embrapa (REGEN) for financial support.

REFERENCES

• ABD-ALLA MH, AL-AMRI SM & EL-ENANY A-WE. 2023. Enhancing Rhizobium–legume symbiosis and reducing nitrogen fertilizer use are potential options for mitigating climate change. Agriculture 13: 2092.
• AKHTAR N, WANI AK, DHANJAL DS & MUKHERJEE S. 2022. Insights into the beneficial roles of dark septate endophytes in plants under challenging environment: resilience to biotic and abiotic stresses. World J Microb Biotech 38: 79.
• ALAM S, KHALIL S, AYUB N & RASHID M. 2002. In vitro solubilization of inorganic phosphate by phosphate solubilizing microorganisms (PSM) from maize rhizosphere. Int J Agri Biol 4: 454-458.
• ALORI ET, DARE MO & BABALOLA OO. 2017. Microbial inoculants for soil quality and plant health. In: Lichtfouse E (Ed), Sustainable Agriculture Reviews, Cham: Springer International Publishing, p. 281-307.
• AYAD F, MATALLAH-BOUTIBA A, ROUANE-HACENE O, BOUDERBALA M & BOUTIBA Z. 2018. Tolerance of Trichoderma sp. to heavy metals and its antifungal activity in algerian marine environment. J Pure Appl Microbiol 12: 855-870.
• AZEVEDO JL. 2014. Endophytic fungi from Brazilian tropical hosts and their biotechnological applications In: Kharwar RN, Upadhyay RS, Dubey NK & Raghuwanshi R (Eds), Microbial Diversity and Biotechnology in Food Security. New Delhi: Springer India, p. 17-22.
• BADER AN, SALERNO GL, COVACEVICH F & CONSOLO VF. 2020. 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.). J King Saud Univ - Sci 32: 867-873.
• BALOGUN DA, OKE MA, ROCHA-MENESES L, FAWOLE OB & OMOJASOLA PF. 2022. Phosphate solubilization potential of indigenous rhizosphere fungi and their biofertilizer formulations. Agron Res 20: 40-55.
• BARROSO CB & NAHAS E. 2013. Enhanced solubilization of iron and calcium phosphates by Aspergillus niger by the addition of alcohols. Braz Arch Biol Technol 56: 181-189.
• BERTHELOT C, CHALOT M, LEYVAL C & BLAUDEZ D. 2019. From darkness to light: Emergence of the mysterious dark septate endophytes in plant growth promotion and stress alleviation. In: Murphy BR, Doohan FM, Saunders MJ & Hodkinson TR (Eds), Endophytes for a Growing World. Cambridge: Cambridge University Press, p. 143-164.
• BERTHELOT C, LEYVAL C, FOULON J, CHALOT M & BLAUDEZ D. 2016. Plant growth promotion, metabolite production and metal tolerance of dark septate endophytes isolated from metal-polluted poplar phytomanagement sites. FEMS Microb Ecol 92: 1-14.
• BERTHELOT C, PERRIN Y, LEYVAL C & BLAUDEZ D. 2017. Melanization and ageing are not drawbacks for successful agro-transformation of dark septate endophytes. Fung Bio 121: 652-663.
• CHANDRAKER D, SAO S, DESHMUKH YK, VERMA L & SAHU PK. 2014. Isolation of Aspergillus niger from Allium cepa bulb and production of citric acid from it. Int J Pharm Bio Sci 5: 144-147.
• CHOLKE SNEHAL S, MAHALE SUVARNA D, DAREKAR KALYANI P & DAITHANKAR ROHINI N. 2019. Production and optimization of citric acid by Aspergillus niger isolated from rotted onion bulb. Int Res J Eng and Tech 6: 2075-2079.
• COSTA MS, ROLIM MM, SILVA GFD, SIMÕES NETO DE, SANTOS JÚNIOR JA & SILVA ÊFDFE. 2021. Biometric responses of sugarcane under high doses of vinasse. Rev Bras Eng Agríc Ambiente 25: 641-647.
• CRUSBERG TC. 2004. Biomineralization of Copper by a Fungus Revealed by SEM. Micr and Anal (UK) 4: 11-13.
• DIGHTON J. 2007. Nutrient cycling by saprotrophic fungi in terrestrial habitats. In: Kubicek CP & Druzhinina IS (Eds), Environmental and Microbial Relationships. Berlin, Heidelberg: Springer Berlin Heidelberg, p. 287-300.
• EDI-PREMONO M, MOAWAD AM & VLECK PLG. 1996. Effect of phosphate solubilizing Pseudomonas putida on the growth of maize and its survival in the rhizosphere. Indonesian J Crop Sci 11: 13-23.
• EISENMAN H & CASADEVALL A. 2011. Synthesis and assembly of fungal melanin. Appl Microbiol and Biotech 93: 931-940.
• EL-GHANDOUR IA, EASA SM, ABDEL AZIZ OA, MOUSA AM & HUSSEIN HG. 2018. Isolation and characterization of phosphate solubilizing fungus in vitro. J Nucl Tech Appl Sci 6: 93-105.
• FARIA PSA, SENÁBIO JA, SOARES MA, SILVA FG, CUNHA APA & SOUCHIE EL. 2016. Assessment of functional traits in the assemblage of endophytic fungi of Anacardium othonianum Rizzini. Pakistan J Bot 48: 1241-1252.
• FERREIRA DF. 2011. Sisvar: a computer statistical analysis system. Cienc Agrotec 35: 1039-1042.
• FISCHER H, MARCONDES DE ALMEIDA JE, LIMBERGER C, HARAKAVA R, SATO ME, COSTA VA, SCHOENINGER K, LEITE LG, CHACON-OROZCO JG & BALDO FB. 2023. The rise of bioinputs in the brazilian agri-industry: trends, cases and hurdles. Outlooks Pest Manag 34: 106-118.
• FOMINA MA, ALEXANDER IJ, COLPAERT JV & GADD GM. 2005. Solubilization of toxic metal minerals and metal tolerance of mycorrhizal fungi. Soil Biol Bioch 37: 851-866.
• FORS RO, PATREZE CM, BERBARA RLL, CARNEIRO MAC & SAGGIN-JÚNIOR OJ. 2020. Dark septate endophytic fungi associated with sugarcane plants cultivated in São Paulo, Brazil. Diversity 12: 351.
• GABER DA, BERTHELOT C, CAMEHL I, KOVÁCS GM, BLAUDEZ D & FRANKEN P. 2020. Salt stress tolerance of dark septate endophytes is independent of melanin accumulation. Front Microbiol 11: 562931.
• GADD GM & DE ROME L. 1988. Biosorption of copper by fungal melanin. Appl Microbiol Biotechnol 29: 610-617.
• GADD GM & SAYER JA. 2000. Fungal transformations of metals and metalloids. In: Lovley DR (Ed), Environmental Microbe –Metal Interactions. Washington: Am Soc Microbiol, p. 237-256.
• GEISEL N, VILAR JMG & RUBI JM. 2011. Optimal resting-growth strategies of microbial populations in fluctuating environments. PLoS ONE 6: e18622.
• GOSWAMI D, PITHWA S, DHANDHUKIA P & THAKKER JN. 2014. Delineating Kocuria turfanensis 2M4 as a credible PGPR: a novel IAA-producing bacteria isolated from saline desert. J Plant Interac 9: 566-576.
• GUILLÉN S, MARCÉN M, FAU E, MAÑAS P & CEBRIÁN G. 2022. Relationship between growth ability, virulence, and resistance to food-processing related stresses in non-typhoidal Salmonellae. Int J Food Microbiol 361: 109462.
• GUPTA NK, SENGUPTA A, GUPTA A, SONAWANE JR & SAHOO H. 2018. Biosorption-an alternative method for nuclear waste management: A critical review. J Environ Chem Eng 6: 2159-2175.
• HAMAYUN M ET AL. 2009. Cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of Glycine max (L.) Merr. World J Microb Biotech 25: 627-632.
• HOU L, LI X, HE X, ZUO Y, ZHANG D & ZHAO L. 2021. Effect of dark septate endophytes on plant performance of Artemisia ordosica and associated soil microbial functional group abundance under salt stress. Appl Soil Ecol 165: 103998.
• HØYER AK, JØRGENSEN HJL, HODKINSON TR & JENSEN B. 2022. Fungal Endophytes Isolated from Elymus repens, a Wild Relative of Barley, Have Potential for Biological Control of Fusarium culmorum and Pyrenophora teres in Barley. Pathogens 25: 1097.
• IDRIS EE, IGLESIAS DJ, TALON M & BORRISS R. 2007. Tryptophan-Dependent Production of Indole-3-Acetic Acid (IAA) Affects Level of Plant Growth Promotion by Bacillus amyloliquefaciens FZB42. MPMI 20: 619-626.
• IRAM S, SHABBIR R, ZAFAR H & JAVAID M. 2015. Biosorption and bioaccumulation of copper and lead by heavy metal-resistant fungal isolates, Arab J Sci Eng 40: 1867-1873.
• IRAM S, ZAMAN A, IQBAL Z & SHABBIR R. 2013. Heavy metal tolerance of fungus isolated from soil contaminated with sewage and industrial wastewater. Polish J Environ Stud 22: 691-697.
• KHAN R, SHAHZAD S, CHOUDHARY M, KHAN S & AHMAD A. 2010. Communities of endophytic fungi in medicinal plant Withania somnifera. Pak J Bot 42: 1281-1287.
• KHATER AEM. 2008. Uranium and heavy metals in Phosphate Fertilizers. In: Merkel BJ & Hasche-Berger A (Eds), Uranium, Mining and Hydrogeology. Berlin, Heidelberg: Springer Berlin Heidelberg, p. 193-198.
• KOCHAR M & SRIVASTAVA S. 2012. Surface colonization by Azospirillum brasilense SM in the indole-3-acetic acid dependent growth improvement of sorghum. J Basic Microbiol 52: 123-131.
• KOREJO F, ALI SA, SHAFIQUE H, VIQAR S, ARA J & EHTESHAMUL-HAQUE S. 2014. Antifungal and antibacterial activity of endophytic Penicillium species isolated from Salvadora species. Pak J Bot 46: 2313-2318.
• KOUR D, RANA KL, KAUR T, YADAV N, YADAV AN, KUMAR M, KUMAR V, DHALIWAL HS & SAXENA AK. 2021. Biodiversity, current developments and potential biotechnological applications of phosphorus-solubilizing and -mobilizing microbes: A review. Pedosphere 31: 43-75.
• LANFRANCO L, BALSAMO R, MARTINO E, PEROTTO S & BONFANTE P. 2010. Zinc ions alter morphology and chitin deposition in an ericoid fungus. Eur J Histochem 46: 341-350.
• LESTARI D, ASRUL L, KUSWINANTI T, MUSA Y & SULFAHRI. 2021. Selection of fungi that potentially produces IAA (Indole Acetic Acid) hormone origin of Takalar sugar factory waste. IOP Conf Ser: Eart Environ Sci 807: 022039.
• LI T, LIU MJ, ZHANG XT, ZHANG HB, SHA T & ZHAO ZW. 2011. Improved tolerance of maize (Zea mays L.) to heavy metals by colonization of a dark septate endophyte (DSE) Exophiala pisciphila. Sci Total Environ 409: 1069-1074.
• LI Z, BAI T, DAI L, WANG F, TAO J, MENG S, HU Y, WANG S & HU S. 2016. A study of organic acid production in contrasts between two phosphate solubilizing fungi: Penicillium oxalicum and Aspergillus niger. Sci Rep 6: 25313.
• MACKIE KA, MARHAN S, DITTERICH F, SCHMIDT HP & KANDELER E. 2015. The effects of biochar and compost amendments on copper immobilization and soil microorganisms in a temperate vineyard. Agric Eco Environ 201: 58-69.
• MAHAMUNI S, WANI P & PATIL A. 2012. Isolation of phosphate solubilizing fungi from rhizosphere of sugarcane & sugar beet using TCP & RP solubilization. Asian J of Biochem and Pharma Res 2: 237-244.
• MANDYAM K & JUMPPONEN A. 2014. Unraveling the dark septate endophyte functions: Insights from the Arabidopsis model. In: Verma VC & Gange AC (Eds), Advances in Endophytic Research. New Delhi: Springer India, p. 115-141.
• MANDYAM KG & JUMPPONEN A. 2015. Mutualism–parasitism paradigm synthesized from results of root-endophyte models. Front Microbiol 5: 776.
• MANDYAM K, LOUGHIN T & JUMPPONEN A. 2010. Isolation and morphological and metabolic characterization of common endophytes in annually burned tallgrass prairie. Mycol 102: 813-821.
• MARTINS MDE & CAMPOS DTDS. 2011. Qualidade microbiológica do solo fertirrigado com vinhaça. Rev Ci Agro-Amb 9: 273-282.
• MENDES GDO, FREITAS ALMD, PEREIRA OL, SILVA IRD, VASSILEV NB & COSTA MD. 2014. Mechanisms of phosphate solubilization by fungal isolates when exposed to different P sources. Ann of Microb 64: 239-249.
• MENDES GDO, VASSILEV NB, BONDUKI VHA, SILVA IRD, RIBEIRO-JR JI & COSTA MD. 2013. Inhibition of Aspergillus niger phosphate solubilization by fluoride released from rock phosphate. App and Environ Microb 79: 4906-4913.
• MOGHADDAM MSH, SAFAIE N, SOLTANI J & HAGH-DOUST N. 2021. Desert-adapted fungal endophytes induce salinity and drought stress resistance in model crops. Pl Physiol Biochem 160: 225-238.
• MUÑOZ R, ALVAREZ MT, MUÑOZ A, TERRAZAS E, GUIEYSSE B & MATTIASSON B. 2006. Sequential removal of heavy metals ions and organic pollutants using an algal-bacterial consortium. Chemosp 63: 903-911.
• NARSIAN V & PATEL HH. 2000. Aspergillus aculeatus as a rock phosphate solubilizer. Soil Biol and Biochem 32: 559-565.
• NAUREEN A, NASIM FH, CHOUDHARY MS, ASHRAF M, GRUNDLER FMW & SCHLEKER ASS. 2022. A new endophytic fungus CJAN1179 isolated from the Cholistan desert promotes lateral root growth in Arabidopsis and produces IAA through tryptophan-dependent pathway. Arch Microbiol 204: 181.
• O’CALLAGHAN M, BALLARD RA & WRIGHT D. 2022. Soil microbial inoculants for sustainable agriculture: Limitations and opportunities. Soil Use Manag 38: 1340-1369.
• OLADIPO OG, AWOTOYE OO, OLAYINKA A, BEZUIDENHOUT CC & MABOETA MS. 2018. Heavy metal tolerance traits of filamentous fungi isolated from gold and gemstone mining sites. Braz J Microb 49: 29-37.
• OLIVEIRA AGD, CHAGAS-JUNIOR AF, SANTOS GRD, MILLER LO & CHAGAS LFB. 2012. Potencial de solubilização de fosfato e produção de AIA por Trichoderma spp. Rev V Agroec Desenv Sust 7: 149-155.
• OLIVEIRA AMD, COSTA MRD, GRAZZIOTTI PH, ABREU CMD, BISPO NDS, ROA JPB, SILVA DM & MIRANDA JM. 2022. Brazilian scenario of inoculant production: A look at patents. Rev Bras Ciênc Solo 46: e0210081.
• PIKOVSKAYA I. 1948. Mobilization of phosphorus in soil connection with the vital activity of some microbial species. Microbiol 17: 362-370.
• PONGRAC P, SONJAK S, VOGEL-MIKUŠ K, KUMP P, NEČEMER M & REGVAR M. 2009. Roots of metal hyperaccumulating population of Thlaspi praecox (brassicaceae) harbour arbuscular mycorrhizal and other fungi under experimental conditions. Int J Phytorremediat 11: 347-359.
• POTISEK M, LIKAR M, VOGEL-MIKUŠ K, ARČON I, GRDADOLNIK J & REGVAR M. 2021. 1,8-dihydroxy naphthalene (DHN) - melanin confers tolerance to cadmium in isolates of melanised dark septate endophytes. Ecotoxicol Environ Saf 222: 112493.
• PRADHAN N & SUKLA LB. 2005. Solubilization of inorganic phosphates by fungi isolated from agriculture soil. Afr J Biotech 5: 850-854.
• PRASAD A, DIXIT M, MEENA SK, SUMAN & KUMAR A. 2023. Qualitative and quantitative estimation for phosphate solubilizing ability of Trichoderma isolates: A natural soil health enhancer. Materials Today: Proceedings 81: 360-366.
• PRIYADHARSINI P & MUTHUKUMAR T. 2017. The root endophytic fungus Curvularia geniculata from Parthenium hysterophorus roots improves plant growth through phosphate solubilization and phytohormone production. Fung Ecol 27: 69-77.
• QIANG X, DING J, LIN W, LI Q, XU C, ZHENG Q & LI Y. 2019. Alleviation of the detrimental effect of water deficit on wheat (Triticum aestivum L.) growth by an indole acetic acid-producing endophytic fungus. Pl Soil 439: 373-391.
• RAWAT R & TEWARI L. 2011. Effect of abiotic stress on phosphate solubilization by biocontrol fungus Trichoderma sp. Curr Microbiol 62: 1521-1526.
• RAZA M, ZHANG Z-F, HYDE KD, DIAO Y-Z & CAI L. 2019. Culturable plant pathogenic fungi associated with sugarcane in southern China. Fung Divers 99: 1-104.
• RICHARDSON AE. 2001. Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Funct Pl Biol 28: 897-906.
• RODRIGUES EP, OLIVEIRA ALM, VIDAL MS, SIMÕES-ARAUJO JL & BALDANI JI. 2007. Obtenção e seleção de mutantes Tn5 de Gluconacetobacter diazotrophicus (Pal 5) com alterações na produção de auxinas. Seropédica: Embrapa Agrobiologia. Bol Pesq Desenvolv 27: 24.
• ROJAS CONTRERAS A, RODRÍGUEZ DORANTES AM, MONTES VILLAFÁN S, PÉREZ JIMÉNEZ S, RODRÍGUEZ TOVAR A & GUERRERO ZÚÑIGA LA. 2010. Evaluación de la promoción del crecimiento de Cynodon dactylon L. por rizobacterias productoras de fitohormonas aisladas de un suelo contaminado con hidrocarburos derivados del petróleo. Polibotánica 29: 131-147.
• RUIZ BERRAQUERO F, BAYA AM & RAMOS CORMENZANA A. 1976. Establecimiento de índices para el estudio de la solubilización de fosfatos por bacterias del suelo. Ars 17: 399-406.
• SARWAR M & KREMER RJ. 1995. Determination of bacterially derived auxins using a microplate method. Let in App Microbiol 20: 282-285.
• SCOTT AJ & KNOTT M. 1974. A Cluster Analysis Method for Grouping Means in the Analysis of Variance. Biomet 30: 507-512.
• SERENO ML, ALMEIDA RS, NISHIMURA DS & FIGUEIRA A. 2007. Response of sugarcane to increasing concentrations of copper and cadmium and expression of metallothionein genes. J Pl Physiol 164: 1499-1515.
• SHADMANI L, JAMALI S & FATEMI A. 2021. Isolation, identification, and characterization of cadmium-tolerant endophytic fungi isolated from barley (Hordeum vulgare L.) roots and their role in enhancing phytoremediation. Braz J Microbiol 52: 1097-1106.
• SILVA APM, BONO JAM & PEREIRA FAR. 2014. Aplicação de vinhaça na cultura da cana-de-açúcar: Efeito no solo e na produtividade de colmos. Rev Bras Eng Agríc Ambient 18: 38-43.
• SILVA-FILHO GN & VIDOR C. 2000. Solubilização de fosfatos por microrganismos na presença de fontes de carbono. Rev Bras Ciênc Solo 24: 311-319.
• SILVA GDC, KITANO IT, RIBEIRO IADF & LACAVA PT. 2022. The potential use of actinomycetes as microbial inoculants and biopesticides in agriculture. Front Soil Sci 2: 833181.
• SOUCHIE EL, AZCÓN R, BAREA JM, SAGGIN-JÚNIOR OJ & SILVA EMRD. 2005. Solubilização de fosfatos em meios sólido e líquido por bactérias e fungos do solo. Pesq Agrop Bras 40: 1149-1152.
• SOUCHIE EL, AZCÓN R, BAREA JM, SAGGIN-JÚNIOR OJ & SILVA EMRD. 2007. Indolacetic acid production by P-solubilizing microorganisms and interaction with arbuscular mycorrhizal fungi. Acta Scient Biol Sci 29: 315-320.
• SOUZA RD, AMBROSINI A & PASSAGLIA LMP. 2015. Plant growth-promoting bacteria as inoculants in agricultural soils. Genet Mol Biol 38: 401-419.
• SU Z-Z, DAI M-D, ZHU J-N, LIU X-H, LI L, ZHU X-M, WANG J-Y, YUAN Z-L & LIN F-C. 2021. Dark septate endophyte Falciphora oryzae-assisted alleviation of cadmium in rice. J Hazard Mater 419: 126435.
• TURBAT A ET AL. 2020. Characterization of the plant growth-promoting activities of endophytic fungi isolated from Sophora flavescens. Microorg 8: 683.
• VERGARA C, ARAUJO KEC, MENEZES-JÚNIOR IDA & ZILLI JÉ. 2019. Caracterização morfofisiológica de isolados de fungos “dark septate”. In: Silva-Matos RRSD, Souza GMMD & Costa ACS (Eds), As ciências biológicas nas dimensões humanista, crítica e reflexiva, Ponta Grossa: Atena Editora, p. 124-135.
• WANG H-Y, LIU S, ZHAI L-M, ZHANG J-Z, REN T-Z, FAN B-Q & LIU H-B. 2015. Preparation and utilization of phosphate biofertilizers using agricultural waste. J Integ Agric 14: 158-167.
• WANG Y, LI P, ZHANG B, WANG Y, MENG J, GAO Y, HE X & HU X. 2020. Identification of phosphate-solubilizing microorganisms and determination of their phosphate-solubilizing activity and growth-promoting capability. BioRes 15: 2560-2578.
• WAQAS M, KHAN AL, KAMRAN M, HAMAYUN M, KANG SM, KIM YH & LEE IJ. 2012. Endophytic fungi produce gibberellins and indoleacetic acid and promotes host-plant growth during stress. Molecul 17: 10754-10773.
• WHITELAW MA, HARDEN TJ & HELYAR KR. 1999. Phosphate solubilisation in solution culture by the soil fungus Penicillium radicum. Soil Biol Biochem 31: 655-665.
• YAKTI W, KOVÁCS GM, VÁGI P & FRANKEN P. 2018. Impact of dark septate endophytes on tomato growth and nutrient uptake. Pl Ecol & Divers 11: 637-648.
• YUNG L, BLAUDEZ D, MAURICE N, AZOU-BARRÉ A & SIRGUEY C. 2021. Dark septate endophytes isolated from non-hyperaccumulator plants can increase phytoextraction of Cd and Zn by the hyperaccumulator Noccaea caerulescens. Environ Sci Pollut Res Int 28: 16544-16557.
• ZAFAR S, AQIL F & AHMAD I. 2007. Metal tolerance and biosorption potential of filamentous fungi isolated from metal contaminated agricultural soil. Biores Techn 98: 2557-2561.
• ZAPOTOCZNY S, JURKIEWICZ A, TYLKO G, ANIELSKA T & TURNAU K. 2007. Accumulation of copper by Acremonium pinkertoniae, a fungus isolated from industrial wastes. Microbiol Res 162: 219-228.
• ZENG Q, LING Q, HU F, WU J, YANG Z, QI Y & LI Q. 2017. Genotypic differences in growth and antioxidant enzyme activities under cadmium stress in sugarcane. Bull Environ Cont Toxicol 99: 607-613.
• ZHANG Y, LI T & ZHAO Z-W. 2013. Colonization characteristics and composition of dark septate endophytes (DSE) in a lead and zinc slag heap in southwest China. Soil Sed Contamin Int J 22: 532-545.
• ZHANG Y, ZHANG Y, LIU M, SHI X & ZHAO Z. 2008. Dark septate endophyte (DSE) fungi isolated from metal polluted soils: Their taxonomic position, tolerance, and accumulation of heavy metals in vitro. J Microbiol 46: 624-632.
• ZHU L, LI T, WANG C, ZHANG X, XU L, XU R & ZHAO Z. 2018. The effects of dark septate endophyte (DSE) inoculation on tomato seedlings under Zn and Cd stress. Environ Sci Pollut Res Int 25: 35232-35241.

Appendix 1

Table AI. Mean Colony Diameter (MCD), Mean Halo Diameter (MHD), and Solubilization Index (SI) of colonies from different strains of Periconia macrospinosa after incubation for 48 and 72 hours on Pikovskaya agar (PVK) medium with calcium phosphate. Averages in descending order.

Culture for 48 hours										Culture for 72 hours
Strain	MCD (mm)		Strain	MHD (mm)		Strain	SI			Strain	MCD (mm)		Strain	MHD (mm)		Strain	SI
A. niger	31.25	a	A. niger	27.29	a	A356	2.16	a		A. niger	47.80	a	A. niger	42.22	a	A356	2.02	a
A333	19.81	b	A155	13.14	b	A155	2.09	a		A161	29.35	b	A356	16.22	b	A. niger	1.88	b
A257	19.01	b	A356	12.02	b	A316	1.97	b		A333	28.73	b	A155	15.01	b	A327	1.83	c
A236	17.77	c	A327	11.08	c	A327	1.93	b		A226	27.02	c	A327	13.05	c	A155	1.75	d
A226	17.75	c	A423	10.84	c	A235	1.91	b		A258	27.01	c	A316	13.00	c	A235	1.75	d
A337	17.75	c	A260	10.58	c	A. niger	1.87	b		A190	26.93	c	A260	12.78	c	A162	1.70	d
A335	17.53	c	A336	10.44	c	A157	1.76	c		A236	26.67	c	A423	12.74	c	A316	1.63	e
A191	17.45	c	A316	10.25	d	A423	1.74	c		A335	26.38	d	A336	12.44	c	A336	1.63	e
A499	17.10	c	A257	9.78	d	A336	1.73	c		A315	26.16	d	A235	12.32	c	A423	1.63	e
A260	16.70	d	A235	9.71	d	A162	1.71	c		A317	26.08	d	A257	11.97	d	A260	1.55	f
A190	16.62	d	A317	9.67	d	A154	1.70	c		A337	26.07	d	A317	11.65	d	A157	1.55	f
A156	16.57	d	A154	9.57	d	A317	1.66	c		A338	26.06	d	A157	11.55	d	A330	1.53	f
A189	16.42	d	A157	9.51	d	A260	1.64	c		A237	26.05	d	A158	11.46	d	A158	1.50	g
A224	16.39	d	A158	9.46	d	A158	1.63	c		A154	25.67	d	A162	11.42	d	A257	1.48	g
A315	16.35	d	A337	9.34	d	A238	1.59	d		A257	25.56	d	A238	11.08	d	A238	1.48	g
A424	15.90	d	A156	8.97	e	A156	1.54	d		A424	25.46	d	A154	10.91	d	A317	1.45	g
A160	15.86	d	A162	8.82	e	A337	1.53	d		A156	25.29	d	A337	10.69	d	A154	1.42	h
A258	15.84	d	A238	7.88	e	A257	1.53	d		A334	25.00	d	A156	9.78	e	A337	1.41	h
A328	15.71	d	A258	1.71	f	A258	1.12	e		A159	24.65	d	A258	9.65	e	A224	1.39	h
A159	15.62	d	A224	1.46	f	A224	1.08	e		A318	24.63	d	A330	9.61	e	A156	1.39	h
A161	15.55	d	A111	0.00	f	A111	1.00	e		A224	24.33	d	A224	9.51	e	A258	1.36	h
A111	15.53	d	A113	0.00	f	A113	1.00	e		A191	24.16	d	A111	0.00	f	A111	1.00	i
A317	15.36	d	A115	0.00	f	A115	1.00	e		A328	23.96	d	A113	0.00	f	A113	1.00	i
A318	15.34	d	A125	0.00	f	A125	1.00	e		A372	23.54	e	A115	0.00	f	A115	1.00	i
A372	15.15	d	A126	0.00	f	A126	1.00	e		A238	23.38	e	A125	0.00	f	A125	1.00	i
A158	15.10	d	A159	0.00	f	A159	1.00	e		A189	23.33	e	A126	0.00	f	A126	1.00	i
A355	14.90	d	A160	0.00	f	A160	1.00	e		A158	23.18	e	A159	0.00	f	A159	1.00	i
A423	14.85	d	A161	0.00	f	A161	1.00	e		A260	23.17	e	A160	0.00	f	A160	1.00	i
A334	14.67	d	A163	0.00	f	A163	1.00	e		A314	23.02	e	A161	0.00	f	A161	1.00	i
A336	14.38	e	A164	0.00	f	A164	1.00	e		A160	22.99	e	A163	0.00	f	A163	1.00	i
A259	14.31	e	A189	0.00	f	A189	1.00	e		A355	22.98	e	A164	0.00	f	A164	1.00	i
A447	14.24	e	A190	0.00	f	A190	1.00	e		A115	22.79	e	A189	0.00	f	A189	1.00	i
A113	14.24	e	A191	0.00	f	A191	1.00	e		A225	22.75	e	A190	0.00	f	A190	1.00	i
A164	14.21	e	A225	0.00	f	A225	1.00	e		A302	22.59	e	A191	0.00	f	A191	1.00	i
A225	14.06	e	A226	0.00	f	A226	1.00	e		A111	22.46	e	A225	0.00	f	A225	1.00	i
A237	13.82	e	A236	0.00	f	A236	1.00	e		A331	22.29	e	A226	0.00	f	A226	1.00	i
A154	13.65	e	A237	0.00	f	A237	1.00	e		A164	22.27	e	A236	0.00	f	A236	1.00	i
A115	13.35	e	A259	0.00	f	A259	1.00	e		A447	22.03	e	A237	0.00	f	A237	1.00	i
A238	13.30	e	A302	0.00	f	A302	1.00	e		A373	21.91	f	A259	0.00	f	A259	1.00	i
A331	13.28	e	A314	0.00	f	A314	1.00	e		A113	21.76	f	A302	0.00	f	A302	1.00	i
A302	13.15	e	A315	0.00	f	A315	1.00	e		A499	21.67	f	A314	0.00	f	A314	1.00	i
A157	13.08	e	A318	0.00	f	A318	1.00	e		A157	21.60	f	A315	0.00	f	A315	1.00	i
A373	13.07	e	A328	0.00	f	A328	1.00	e		A259	21.35	f	A318	0.00	f	A318	1.00	i
A163	12.91	e	A330	0.00	f	A330	1.00	e		A163	21.28	f	A328	0.00	f	A328	1.00	i
A162	12.52	f	A331	0.00	f	A331	1.00	e		A126	21.09	f	A331	0.00	f	A331	1.00	i
A314	12.32	f	A332	0.00	f	A332	1.00	e		A423	20.93	f	A332	0.00	f	A332	1.00	i
A155	12.09	f	A333	0.00	f	A333	1.00	e		A336	20.88	f	A333	0.00	f	A333	1.00	i
A327	11.95	f	A334	0.00	f	A334	1.00	e		A316	20.55	f	A334	0.00	f	A334	1.00	i
A338	11.90	f	A335	0.00	f	A335	1.00	e		A125	20.54	f	A335	0.00	f	A335	1.00	i
A332	11.49	f	A338	0.00	f	A338	1.00	e		A332	20.20	f	A338	0.00	f	A338	1.00	i
A330	11.37	f	A355	0.00	f	A355	1.00	e		A155	19.98	f	A355	0.00	f	A355	1.00	i
A125	11.08	f	A372	0.00	f	A372	1.00	e		A330	18.39	g	A372	0.00	f	A372	1.00	i
A316	10.92	f	A373	0.00	f	A373	1.00	e		A356	16.56	g	A373	0.00	f	A373	1.00	i
A235	10.76	f	A424	0.00	f	A424	1.00	e		A162	16.54	g	A424	0.00	f	A424	1.00	i
A126	10.74	f	A447	0.00	f	A447	1.00	e		A235	16.42	g	A447	0.00	f	A447	1.00	i
A356	10.68	f	A499	0.00	f	A499	1.00	e		A327	15.69	g	A499	0.00	f	A499	1.00	i

Means followed by the same letter in the columns do not differ by the Scott-Knott 5% test. A. niger : Aspergillus niger strain A521 as a positive control of solubilization.

Table AII. Mean colony diameter and colony surface of different strains of Periconia macrospinosa after 96 hours of growth on malt extract-agar (MEA) medium with levels of 0, 400, and 500 mg.L^-1 of cadmium. Averages in descending order.

Mean colony diameter (mm)										Colony surface (cm2)
Strains	0		Strains	400		Strains	500			Strains	0		Strains	400		Strains	500
A331	51.8	a	A226	36.2	a	A226	32.6	a		A331	20.8	a	A226	10.3	a	A226	8.4	a
A335	47.6	b	A423	32.4	b	A332	31.1	a		A335	20.3	a	A423	8.2	b	A332	7.6	a
A226	45.5	b	A332	31.9	b	A157	31.0	a		A226	16.2	b	A332	8.0	b	A157	7.5	a
A318	44.8	b	A158	29.8	c	A423	29.8	a		A318	15.8	b	A158	7.0	b	A423	7.0	a
A237	44.1	b	A160	29.5	c	A328	27.2	b		A237	15.3	b	A160	6.8	b	A328	5.8	b
A157	44.0	b	A161	28.6	c	A158	26.8	b		A157	15.2	b	A161	6.4	c	A158	5.6	b
A160	42.6	c	A328	28.5	c	A161	26.4	b		A160	14.3	b	A328	6.4	c	A161	5.5	b
A424	42.4	c	A159	28.0	c	A160	26.1	b		A424	14.1	b	A159	6.2	c	A160	5.4	b
A330	42.1	c	A355	28.0	c	A318	25.4	c		A330	14.0	b	A355	6.1	c	A318	5.1	b
A154	42.1	c	A318	27.5	c	A355	25.2	c		A154	13.9	b	A318	5.9	c	A355	5.0	b
A314	42.0	c	A164	27.4	c	A424	24.9	c		A314	13.8	b	A164	5.9	c	A424	4.9	b
A328	41.9	c	A157	26.8	c	A447	24.8	c		A328	13.8	b	A157	5.6	c	A447	4.8	b
A156	41.9	c	A314	26.6	c	A159	24.8	c		A156	13.8	b	A314	5.5	c	A159	4.8	b
A332	41.6	c	A156	26.4	c	A191	24.2	c		A329	13.5	c	A156	5.5	c	A191	4.6	b
A191	41.5	c	A162	26.1	d	A336	24.1	c		A332	13.5	c	A162	5.3	c	A336	4.6	b
A238	41.3	c	A237	26.0	d	A162	24.0	c		A191	13.5	c	A237	5.3	c	A162	4.5	b
A315	41.3	c	A155	25.7	d	A237	23.8	c		A315	13.4	c	A155	5.2	c	A237	4.5	b
A447	41.2	c	A424	25.3	d	A337	23.5	c		A238	13.4	c	A424	5.0	c	A327	4.4	b
A329	41.0	c	A336	25.1	d	A238	23.2	c		A447	13.3	c	A330	5.0	c	A337	4.3	b
A317	40.9	c	A330	24.8	d	A327	23.0	c		A317	13.2	c	A336	4.9	c	A238	4.2	b
A158	40.8	c	A337	24.3	d	A314	22.6	d		A158	13.1	c	A327	4.7	c	A314	4.0	c
A302	40.5	c	A259	24.3	d	A259	22.4	d		A302	12.9	c	A337	4.7	c	A259	3.9	c
A159	39.7	d	A327	23.9	d	A156	22.1	d		A159	12.4	c	A259	4.6	c	A156	3.8	c
A316	39.7	d	A315	23.6	d	A225	22.0	d		A316	12.4	c	A447	4.4	c	A225	3.8	c
A423	39.5	d	A373	23.5	d	A190	21.8	d		A423	12.3	c	A315	4.4	c	A190	3.7	c
A327	39.4	d	A447	23.5	d	A338	21.6	d		A327	12.2	c	A373	4.4	c	A338	3.7	c
A161	39.3	d	A257	23.5	d	A257	21.6	d		A164	12.1	c	A257	4.3	c	A164	3.7	c
A164	39.3	d	A235	23.3	d	A164	21.6	d		A161	12.1	c	A235	4.3	c	A257	3.7	c
A259	39.1	d	A238	23.0	d	A373	21.5	d		A259	12.0	c	A238	4.2	c	A373	3.6	c
A257	38.3	d	A258	22.7	d	A224	21.1	d		A257	11.5	c	A258	4.0	d	A224	3.5	c
A373	38.1	d	A338	22.6	d	A126	21.0	d		A373	11.4	d	A338	4.0	d	A126	3.5	c
A190	37.7	d	A372	22.2	d	A333	21.0	d		A190	11.2	d	A372	3.9	d	A333	3.5	c
A258	37.5	d	A225	22.1	d	A235	20.9	d		A258	11.0	d	A225	3.9	d	A235	3.5	c
A372	36.8	e	A191	22.0	d	A258	20.8	d		A372	10.6	d	A191	3.8	d	A258	3.4	c
A225	36.3	e	A190	21.8	d	A315	20.6	d		A225	10.4	d	A190	3.7	d	A315	3.3	c
A113	36.1	e	A260	21.7	d	A330	20.0	d		A113	10.2	d	A260	3.7	d	A260	3.1	c
A111	36.0	e	A224	21.2	d	A260	20.0	d		A111	10.2	d	A224	3.5	d	A330	3.1	c
A337	35.8	e	A333	20.7	d	A334	19.9	d		A155	10.1	d	A333	3.4	d	A334	3.1	c
A162	35.7	e	A334	19.7	e	A372	19.7	d		A337	10.1	d	A334	3.1	d	A372	3.1	c
A189	35.7	e	A189	18.8	e	A189	19.1	e		A162	10.0	d	A189	2.8	d	A189	2.9	c
A224	35.4	e	A113	18.7	e	A113	19.1	e		A189	10.0	d	A113	2.7	d	A113	2.8	c
A155	35.4	e	A111	18.5	e	A111	19.0	e		A224	9.9	d	A111	2.7	d	A111	2.8	c
A355	35.3	e	A126	17.7	e	A356	17.7	e		A355	9.8	d	A126	2.4	e	A356	2.5	d
A260	35.0	e	A124	17.2	e	A317	17.6	e		A336	9.6	d	A124	2.3	e	A317	2.4	d
A235	34.6	e	A115	17.0	e	A124	17.5	e		A260	9.6	d	A115	2.3	e	A124	2.4	d
A336	34.2	f	A317	16.9	e	A115	17.4	e		A235	9.4	d	A317	2.2	e	A115	2.4	d
A124	33.2	f	A154	16.0	f	A163	17.2	e		A124	8.6	d	A154	2.0	e	A163	2.3	d
A338	33.2	f	A125	15.6	f	A302	16.7	e		A338	8.6	d	A356	1.9	e	A302	2.2	d
A115	33.1	f	A356	15.6	f	A316	16.3	e		A115	8.6	d	A125	1.9	e	A316	2.1	d
A126	32.7	f	A316	15.6	f	A154	16.1	e		A126	8.5	d	A316	1.9	e	A154	2.0	d
A236	31.9	f	A302	15.5	f	A125	16.1	e		A236	8.0	e	A302	1.9	e	A125	2.0	d
A356	31.9	f	A499	14.9	f	A499	15.1	e		A356	8.0	e	A499	1.8	e	A499	1.8	d
A125	30.4	g	A163	14.4	f	A236	13.2	f		A125	7.3	e	A163	1.6	e	A155	1.5	d
A499	29.6	g	A236	13.7	f	A155	12.5	f		A499	7.0	e	A236	1.5	e	A236	1.4	d
A333	27.1	h	A331	8.0	g	A335	8.0	g		A333	5.8	f	A331	0.5	e	A335	0.5	d
A334	25.8	h	A335	8.0	g	A329	8.0	g		A334	5.2	f	A335	0.5	e	A329	0.5	d
A163	24.0	h	A329	8.0	g	A331	8.0	g		A163	4.6	f	A329	0.5	e	A331	0.5	d

Means followed by the same letter in the columns do not differ by the Scott-Knott 5% test. The other strains treatments.

Table AIII. Mean colony diameter and colony surface of different strains of Periconia macrospinosa after 120 hours of growth on malt extract-agar (MEA) medium with levels of 0, 300, and 1000 mg.L^-1 of copper. Averages in descending order.

Mean colony diameter (mm)										Colony surface (cm2)
Strains	0		Strains	300		Strains	1000			Strains	0		Strains	300		Strains	1000
A158	56.6	a	A162	44.7	a	A160	34.3	a		A158	25.0	a	A162	15.8	a	A160	9.3	a
A237	55.9	a	A334	44.1	a	A191	31.4	a		A237	24.6	a	A334	15.3	a	A191	7.8	a
A334	55.5	a	A191	44.0	a	A316	31.3	a		A334	24.2	a	A191	15.2	a	A316	7.7	a
A355	55.3	a	A257	42.4	a	A158	31.3	a		A317	24.0	a	A257	14.2	b	A158	7.7	a
A317	55.3	a	A423	41.4	b	A330	31.2	a		A355	24.0	a	A423	13.5	b	A330	7.6	a
A318	55.0	a	A314	41.3	b	A162	30.9	a		A318	23.8	a	A314	13.4	b	A162	7.5	a
A258	54.9	a	A373	41.2	b	A334	30.4	a		A258	23.6	a	A373	13.3	b	A334	7.5	a
A257	54.8	a	A164	40.9	b	A499	30.1	a		A257	23.6	a	A164	13.2	b	A499	7.4	a
A314	53.6	a	A499	40.5	b	A257	29.4	b		A314	22.6	b	A499	13.1	b	A257	6.9	b
A226	53.1	b	A317	40.4	b	A423	29.3	b		A226	22.2	b	A317	13.0	b	A423	6.8	b
A191	53.1	b	A259	39.9	b	A329	29.1	b		A191	22.1	b	A259	12.5	b	A329	6.7	b
A162	53.1	b	A157	39.0	b	A190	28.8	b		A162	22.1	b	A332	12.0	c	A238	6.7	b
A315	53.0	b	A332	38.8	b	A336	28.8	b		A315	22.0	b	A157	11.9	c	A226	6.6	b
A160	52.6	b	A190	38.3	c	A226	28.7	b		A160	21.7	c	A190	11.5	c	A190	6.6	b
A159	52.5	b	A333	37.9	c	A238	28.6	b		A159	21.6	c	A333	11.3	c	A336	6.5	b
A373	52.5	b	A158	37.2	c	A164	28.4	b		A373	21.6	c	A158	10.9	c	A164	6.3	b
A336	52.3	b	A154	37.1	c	A424	28.2	b		A336	21.4	c	A154	10.8	c	A424	6.3	b
A332	52.2	b	A258	36.3	c	A314	27.9	b		A332	21.4	c	A258	10.3	c	A314	6.1	b
A499	51.9	b	A160	36.0	c	A125	27.8	b		A499	21.2	c	A160	10.3	c	A125	6.1	b
A260	51.8	b	A318	35.8	c	A337	27.8	b		A260	21.1	c	A318	10.1	c	A337	6.1	b
A259	51.6	b	A155	35.6	c	A333	27.8	b		A259	20.9	c	A238	9.9	c	A333	6.1	b
A157	51.1	b	A238	35.4	c	A259	27.7	b		A157	20.5	c	A155	9.9	c	A259	6.1	b
A302	50.8	b	A355	34.8	c	A159	26.0	c		A302	20.3	c	A355	9.6	c	A159	5.4	b
A238	50.4	b	A315	34.5	c	A356	25.9	c		A238	20.0	d	A315	9.3	d	A356	5.4	b
A224	49.9	b	A224	34.1	c	A225	25.7	c		A224	19.5	d	A224	9.1	d	A225	5.2	b
A316	49.8	b	A328	33.9	c	A327	25.1	c		A316	19.4	d	A328	9.1	d	A318	5.0	b
A338	49.1	c	A226	33.8	c	A163	24.9	c		A338	18.9	d	A226	9.0	d	A327	5.0	b
A189	48.8	c	A337	32.8	d	A318	24.7	c		A189	18.7	d	A330	8.5	d	A163	4.9	b
A447	48.4	c	A189	32.5	d	A373	23.9	c		A447	18.4	d	A337	8.4	d	A373	4.5	c
A372	47.8	c	A330	32.4	d	A126	23.1	d		A372	18.0	e	A189	8.3	d	A189	4.4	c
A330	47.5	c	A159	32.4	d	A111	22.8	d		A330	17.8	e	A159	8.2	d	A126	4.2	c
A423	47.4	c	A125	32.3	d	A189	22.3	d		A156	17.5	e	A125	8.2	d	A111	4.1	c
A156	47.2	c	A329	32.1	d	A235	22.1	d		A423	17.4	e	A329	8.1	d	A302	3.9	c
A155	46.7	c	A336	32.0	d	A302	21.9	d		A155	17.1	e	A336	8.1	d	A235	3.8	c
A154	46.7	c	A327	30.8	d	A372	21.2	d		A154	17.1	e	A327	7.5	e	A372	3.5	c
A333	46.5	c	A260	30.8	d	A447	20.9	d		A333	16.9	e	A260	7.4	e	A447	3.5	c
A236	46.2	c	A156	30.8	d	A236	20.9	d		A236	16.8	e	A156	7.4	e	A236	3.4	c
A225	46.2	c	A302	30.4	d	A154	20.7	d		A225	16.7	e	A302	7.3	e	A154	3.4	c
A125	45.8	d	A236	30.2	d	A332	20.1	e		A125	16.5	e	A236	7.1	e	A332	3.3	c
A337	45.8	d	A335	29.8	d	A328	19.6	e		A337	16.5	e	A335	7.0	e	A315	3.1	c
A424	45.8	d	A237	29.3	d	A315	19.5	e		A424	16.5	e	A237	6.7	e	A260	3.0	c
A329	45.1	d	A316	29.0	d	A224	19.3	e		A327	16.1	f	A316	6.7	e	A328	3.0	c
A327	44.9	d	A447	29.0	d	A260	19.0	e		A329	16.0	f	A447	6.6	e	A224	2.9	c
A331	44.7	d	A372	28.5	e	A157	18.9	e		A335	15.7	f	A372	6.4	e	A157	2.8	d
A124	44.4	d	A356	28.2	e	A156	18.8	e		A161	15.5	f	A356	6.4	e	A156	2.8	d
A161	44.4	d	A338	27.6	e	A258	18.6	e		A124	15.5	f	A338	6.0	f	A258	2.7	d
A190	43.9	d	A235	27.5	e	A115	18.4	e		A190	15.1	f	A235	5.9	f	A115	2.7	d
A235	43.8	d	A115	27.1	e	A237	17.7	e		A331	15.1	f	A115	5.8	f	A237	2.5	d
A115	43.6	d	A225	26.7	e	A124	17.6	e		A235	15.1	f	A424	5.8	f	A124	2.5	d
A335	43.2	d	A161	26.6	e	A155	17.2	f		A115	14.9	f	A225	5.6	f	A355	2.3	d
A328	40.2	e	A424	26.1	e	A355	17.1	f		A328	14.3	f	A161	5.6	f	A155	2.3	d
A126	40.1	e	A126	25.8	e	A335	15.7	f		A126	12.6	g	A126	5.2	f	A335	1.9	d
A356	38.4	e	A111	25.3	e	A161	15.3	f		A356	11.7	g	A111	5.0	f	A317	1.9	d
A111	35.6	f	A124	23.2	e	A317	15.3	f		A111	10.0	h	A124	4.2	f	A161	1.8	d
A164	34.3	f	A113	21.0	f	A338	14.5	f		A164	9.5	h	A113	3.4	g	A338	1.7	d
A113	34.1	f	A163	12.9	g	A113	13.0	g		A113	9.5	h	A163	1.3	h	A113	1.3	d
A163	20.6	g	A331	10.2	g	A331	9.5	g		A163	3.3	i	A331	0.8	h	A331	0.7	d

Means followed by the same letter in the columns do not differ by the Scott-Knott 5% test. The other strains treatments.

Table AIV. Mean colony diameter and colony surface of different strains of Periconia macrospinosa after 96 hours of growth on malt extract-agar (MEA) medium with levels of 0, 400, and 600 mg.L^-1 of zinc. Averages in descending order.

Mean colony diameter (mm)										Colony surface (cm2)
Strains	0		Strains	400		Strains	600			Strains	0		Strains	400		Strains	600
A331	51.8	a	A318	42.3	a	A158	32.7	a		A331	20.8	a	A318	14.0	a	A158	8.4	a
A335	47.6	b	A226	42.2	a	A314	30.5	a		A335	20.3	a	A226	14.0	a	A302	7.7	a
A226	45.5	b	A158	39.9	a	A315	30.2	a		A226	16.2	b	A158	12.5	b	A314	7.4	a
A318	44.8	b	A159	39.5	a	A330	30.0	a		A318	15.8	b	A159	12.2	b	A315	7.1	a
A237	44.1	b	A373	39.0	a	A161	29.9	a		A237	15.3	b	A373	11.9	b	A330	7.1	a
A157	44.0	b	A156	38.6	a	A302	29.0	a		A157	15.2	b	A156	11.7	b	A161	7.1	a
A160	42.6	c	A447	38.2	b	A373	28.6	a		A160	14.3	b	A447	11.5	b	A373	6.4	a
A424	42.4	c	A328	38.0	b	A162	27.2	a		A424	14.1	b	A328	11.4	b	A162	5.8	b
A330	42.1	c	A314	37.9	b	A372	27.0	a		A330	14.0	b	A314	11.3	b	A372	5.7	b
A154	42.1	c	A372	37.8	b	A113	23.2	b		A154	13.9	b	A372	11.2	b	A191	4.6	b
A314	42.0	c	A237	37.2	b	A111	23.0	b		A314	13.8	b	A237	10.9	b	A189	4.3	b
A328	41.9	c	A161	37.1	b	A156	22.4	b		A328	13.8	b	A161	10.8	b	A113	4.2	b
A156	41.9	c	A315	36.5	b	A189	22.1	b		A156	13.8	b	A315	10.4	b	A111	4.2	b
A332	41.6	c	A157	36.2	b	A191	21.9	b		A329	13.5	c	A157	10.4	b	A156	3.9	c
A191	41.5	c	A327	34.1	b	A163	21.8	b		A332	13.5	c	A327	9.4	c	A163	3.7	c
A238	41.3	c	A162	33.8	b	A126	21.4	b		A191	13.5	c	A162	8.9	c	A124	3.6	c
A315	41.3	c	A225	33.1	c	A124	21.3	b		A315	13.4	c	A225	8.6	c	A126	3.6	c
A447	41.2	c	A113	32.8	c	A115	21.1	b		A238	13.4	c	A113	8.5	c	A115	3.5	c
A329	41.0	c	A111	32.7	c	A332	20.4	b		A447	13.3	c	A111	8.4	c	A332	3.3	c
A317	40.9	c	A224	32.2	c	A236	20.1	b		A317	13.2	c	A259	8.3	c	A236	3.2	c
A158	40.8	c	A259	32.0	c	A125	19.4	b		A158	13.1	c	A330	8.2	c	A125	3.0	c
A302	40.5	c	A330	31.8	c	A356	18.3	c		A302	12.9	c	A224	8.2	c	A499	2.7	c
A159	39.7	c	A355	31.5	c	A499	18.2	c		A159	12.4	c	A355	8.0	c	A356	2.7	c
A316	39.7	c	A190	31.2	c	A317	18.1	c		A316	12.4	c	A257	7.9	c	A317	2.6	c
A423	39.5	c	A257	31.1	c	A154	17.2	c		A423	12.3	c	A190	7.7	c	A155	2.5	c
A327	39.4	c	A236	30.8	c	A316	16.9	c		A327	12.2	c	A258	7.5	d	A154	2.3	c
A161	39.3	c	A258	30.3	c	A226	16.4	c		A164	12.1	c	A236	7.4	d	A316	2.2	c
A164	39.3	c	A124	30.2	c	A157	15.4	c		A161	12.1	c	A164	7.3	d	A226	2.1	c
A259	39.1	c	A115	30.1	c	A155	15.1	c		A259	12.0	c	A124	7.2	d	A157	1.9	c
A257	38.3	c	A164	29.4	d	A237	15.0	c		A257	11.5	c	A115	7.1	d	A237	1.8	c
A373	38.1	c	A499	29.2	d	A424	14.9	c		A373	11.4	d	A499	6.7	d	A424	1.8	c
A190	37.7	c	A336	28.9	d	A159	14.8	c		A190	11.2	d	A336	6.6	d	A423	1.7	c
A258	37.5	c	A260	28.6	d	A423	14.8	c		A258	11.0	d	A260	6.6	d	A159	1.7	c
A372	36.8	d	A424	28.2	d	A238	14.4	c		A372	10.6	d	A424	6.3	d	A318	1.6	c
A225	36.3	d	A332	28.2	d	A318	14.4	c		A225	10.4	d	A332	6.2	d	A238	1.6	c
A113	36.1	d	A235	28.1	d	A259	14.3	c		A113	10.2	d	A235	6.2	d	A259	1.6	c
A111	36.0	d	A317	27.8	d	A164	14.2	c		A111	10.2	d	A160	6.2	d	A164	1.6	c
A337	35.8	d	A125	27.7	d	A160	14.1	c		A155	10.1	d	A317	6.0	d	A160	1.6	c
A162	35.7	d	A160	27.5	d	A225	13.6	d		A337	10.1	d	A125	6.0	d	A225	1.5	c
A189	35.7	d	A191	26.7	d	A355	13.5	d		A162	10.0	d	A191	5.6	d	A190	1.4	c
A224	35.4	d	A316	26.5	d	A190	13.5	d		A189	10.0	d	A316	5.5	d	A355	1.4	c
A155	35.4	d	A154	26.4	d	A257	13.5	d		A224	9.9	d	A154	5.5	d	A257	1.4	c
A355	35.3	d	A126	25.9	d	A337	13.3	d		A355	9.8	d	A126	5.3	d	A337	1.4	c
A260	35.0	d	A356	25.7	d	A336	13.1	d		A336	9.6	d	A356	5.2	d	A336	1.4	c
A235	34.6	d	A302	25.7	d	A224	12.7	d		A260	9.6	d	A302	5.1	d	A224	1.3	c
A336	34.2	d	A163	22.1	e	A258	12.6	d		A235	9.4	d	A163	3.9	e	A258	1.3	c
A124	33.2	d	A238	22.0	e	A260	12.6	d		A124	8.6	d	A238	3.8	e	A260	1.3	c
A338	33.2	d	A337	20.5	e	A235	12.1	d		A338	8.6	d	A337	3.4	e	A235	1.2	c
A115	33.1	d	A335	19.7	e	A328	12.1	d		A115	8.6	d	A335	3.0	e	A328	1.2	c
A126	32.7	d	A189	19.6	e	A335	12.0	d		A126	8.5	d	A189	3.0	e	A335	1.1	c
A236	31.9	d	A423	19.3	e	A338	12.0	d		A236	8.0	e	A423	2.9	e	A338	1.1	c
A356	31.9	d	A334	18.7	e	A329	11.3	d		A356	8.0	e	A333	2.9	e	A331	1.0	c
A125	30.4	e	A333	18.5	e	A331	11.2	d		A125	7.3	e	A334	2.8	e	A329	1.0	c
A499	29.6	e	A338	18.3	e	A327	11.1	d		A499	7.0	e	A338	2.7	e	A327	1.0	c
A333	27.1	f	A329	15.3	e	A333	9.3	d		A333	5.8	f	A329	1.8	e	A333	0.7	c
A334	25.8	f	A155	11.9	f	A447	8.9	d		A334	5.2	f	A155	1.4	e	A447	0.6	c
A163	23.5	f	A331	8.0	f	A334	8.4	d		A163	4.4	f	A331	0.5	e	A334	0.6	c

Means followed by the same letter in the columns do not differ by the Scott-Knott 5% test.

Table AIV. Table AV. Mean colony diameter and colony surface of different strains of Periconia macrospinosa after 96 hours of growth on malt extract-agar (MEA) medium with levels of 0, 400, and 800 mL.L^-1 of vinasse. Averages in descending order.

Mean colony diameter (mm)										Colony surface (cm2)
Strains	0		Strains	400		Strains	600			Strains	0		Strains	400		Strains	600
A257	49.8	a	A334	46.4	a	A334	52.8	a		A257	20.1	a	A334	16.8	a	A334	21.9	a
A334	48.6	a	A331	42.4	b	A335	43.5	b		A334	18.4	a	A331	14.1	b	A335	14.7	b
A331	46.4	a	A257	42.2	b	A331	40.7	b		A331	16.8	b	A257	14.0	b	A331	13.0	c
A155	46.0	a	A161	41.2	c	A158	38.9	c		A155	16.6	b	A161	13.4	b	A158	11.9	c
A154	43.7	b	A336	41.2	c	A336	38.8	c		A161	15.1	c	A336	13.3	b	A336	11.9	c
A161	43.7	b	A329	41.0	c	A154	38.0	c		A154	15.0	c	A329	13.2	b	A161	11.4	c
A258	43.5	b	A154	40.6	c	A161	38.0	c		A258	14.9	c	A154	13.0	b	A154	11.4	c
A355	43.0	b	A373	40.6	c	A332	37.7	c		A355	14.6	c	A373	12.9	b	A332	11.1	c
A156	41.8	c	A155	40.6	c	A257	37.1	c		A329	13.7	c	A155	12.9	b	A257	10.8	c
A329	41.7	c	A332	40.5	c	A373	36.8	c		A156	13.7	c	A332	12.9	b	A373	10.6	c
A372	41.7	c	A372	40.3	c	A328	36.4	c		A372	13.7	c	A372	12.8	b	A328	10.4	d
A336	41.7	c	A316	40.3	c	A356	36.2	c		A336	13.7	c	A316	12.7	b	A329	10.4	d
A332	41.0	c	A158	40.2	c	A237	36.1	c		A332	13.2	d	A158	12.7	b	A356	10.3	d
A260	40.4	c	A126	40.1	c	A162	36.0	c		A260	12.8	d	A126	12.6	b	A237	10.3	d
A327	40.1	c	A328	39.8	c	A329	35.7	c		A327	12.6	d	A328	12.4	b	A162	10.2	d
A337	39.3	d	A258	39.6	c	A160	35.6	c		A111	12.3	d	A258	12.3	b	A160	10.0	d
A115	39.2	d	A156	39.0	c	A156	35.6	c		A337	12.1	d	A156	11.9	b	A156	9.9	d
A111	39.1	d	A237	38.8	c	A191	34.8	d		A164	12.1	d	A237	11.8	b	A191	9.5	d
A164	39.0	d	A160	38.8	c	A315	34.6	d		A115	12.1	d	A160	11.8	b	A315	9.4	d
A335	38.7	d	A162	38.7	c	A155	34.5	d		A335	11.9	d	A162	11.8	b	A155	9.4	d
A424	38.6	d	A327	38.6	c	A423	34.4	d		A424	11.7	e	A327	11.7	b	A423	9.3	d
A236	38.4	d	A260	38.5	c	A225	34.3	d		A236	11.6	e	A260	11.6	b	A225	9.3	d
A330	38.3	d	A225	38.1	c	A424	34.1	d		A162	11.5	e	A225	11.4	b	A424	9.1	d
A162	38.2	d	A125	37.7	c	A318	34.0	d		A330	11.5	e	A125	11.1	c	A318	9.1	d
A191	38.1	d	A330	37.6	c	A125	34.0	d		A191	11.4	e	A330	11.1	c	A125	9.1	d
A157	37.8	d	A191	37.3	d	A159	34.0	d		A237	11.2	e	A191	10.9	c	A159	9.1	d
A237	37.8	d	A124	37.2	d	A330	33.9	d		A157	11.2	e	A124	10.9	c	A330	9.0	d
A333	37.4	d	A226	37.0	d	A327	33.9	d		A333	11.1	e	A226	10.8	c	A327	9.0	d
A238	37.4	d	A159	37.0	d	A447	33.8	d		A238	11.0	e	A159	10.8	c	A447	9.0	d
A159	37.1	d	A423	37.0	d	A314	33.7	d		A159	10.8	e	A423	10.7	c	A314	8.9	d
A328	37.0	d	A224	36.8	d	A236	33.4	d		A328	10.8	e	A224	10.7	c	A236	8.7	e
A225	36.6	d	A115	36.7	d	A258	33.2	d		A225	10.5	e	A115	10.6	c	A258	8.7	e
A160	36.5	d	A337	36.6	d	A355	33.1	d		A160	10.5	e	A337	10.5	c	A226	8.7	e
A315	36.1	e	A355	36.6	d	A124	33.1	d		A163	10.3	e	A355	10.5	c	A355	8.6	e
A318	36.0	e	A356	36.3	d	A226	33.1	d		A315	10.2	e	A356	10.4	c	A124	8.6	e
A224	35.9	e	A259	36.1	d	A189	33.0	d		A224	10.2	e	A259	10.2	c	A189	8.6	e
A163	35.5	e	A236	36.0	d	A372	32.9	d		A318	10.2	e	A236	10.1	c	A372	8.5	e
A226	35.4	e	A238	35.9	d	A157	32.8	d		A226	9.8	e	A238	10.1	c	A157	8.5	e
A423	35.1	e	A189	35.9	d	A316	32.6	d		A423	9.8	e	A189	10.1	c	A316	8.3	e
A447	34.7	e	A315	35.7	d	A259	32.5	d		A113	9.4	e	A315	10.0	c	A259	8.3	e
A113	34.7	e	A447	35.4	d	A333	32.5	d		A447	9.4	e	A447	9.9	c	A333	8.3	e
A124	34.4	e	A333	35.4	d	A499	32.3	d		A124	9.3	e	A333	9.8	c	A224	8.2	e
A499	33.9	e	A318	35.3	d	A224	32.2	d		A499	9.2	e	A318	9.8	c	A499	8.2	e
A356	33.8	e	A157	35.1	d	A238	32.0	d		A356	9.0	e	A157	9.7	c	A238	8.1	e
A189	33.6	e	A111	35.1	d	A337	31.8	d		A189	8.9	e	A111	9.7	c	A337	8.0	e
A302	32.9	e	A424	35.0	d	A302	31.0	d		A302	8.5	f	A424	9.6	c	A302	7.7	e
A235	31.7	f	A164	34.9	d	A115	30.9	d		A235	7.9	f	A164	9.6	c	A317	7.5	e
A158	31.1	f	A335	34.1	d	A317	30.9	d		A158	7.6	f	A335	9.4	c	A115	7.5	e
A259	31.0	f	A302	33.6	d	A113	30.0	e		A259	7.6	f	A302	8.9	d	A113	7.1	f
A373	30.9	f	A317	33.3	e	A126	29.7	e		A373	7.5	f	A317	8.7	d	A126	6.9	f
A190	30.6	f	A113	33.0	e	A260	29.1	e		A190	7.3	f	A113	8.6	d	A260	6.7	f
A125	29.6	f	A314	32.8	e	A338	28.2	e		A126	6.9	f	A499	8.5	d	A338	6.3	f
A126	29.3	f	A499	32.7	e	A111	26.9	f		A125	6.9	f	A314	8.4	d	A163	6.2	f
A314	28.2	g	A338	31.5	e	A235	26.4	f		A314	6.3	g	A338	7.8	d	A111	5.7	f
A338	27.8	g	A235	30.1	f	A164	26.4	f		A338	6.0	g	A163	7.5	d	A235	5.5	f
A317	26.0	g	A163	29.3	f	A163	25.2	f		A317	5.4	g	A235	7.1	d	A164	5.5	f
A316	25.7	g	A190	25.5	g	A190	23.4	f		A316	5.2	g	A190	5.1	e	A190	4.3	f

Means followed by the same letter in the columns do not differ by the Scott-Knott 5% test.

Table AVI. Indoleacetic acid (IAA) concentration in the culture medium, fresh mycelium mass and IAA produced by mycelium mass of Periconia macrospinosa strains after 120 h of growth in liquid malt extract supplemented with L-tryptophan. Averages in descending order.

Strains	IAA concentration in the medium (µg.mL-1)			Strains	Fresh mycelium mass (g)			Strains	IAA produced by the mycelium (µg.g-1)
A302	8.29	a		A157	0.67	a		A257	638.17	a
A226	7.93	a		A156	0.64	a		A226	567.41	b
A355	7.01	b		A158	0.64	a		A225	500.77	c
A225	6.96	b		A115	0.61	b		A260	483.25	c
A499	6.84	b		A125	0.60	b		A237	459.33	c
A257	6.66	b		A113	0.57	b		A259	446.41	c
A189	6.45	c		A126	0.57	b		A302	413.73	d
A447	6.21	c		A155	0.54	c		A258	384.87	d
A333	5.94	c		A159	0.54	c		A499	372.00	d
A337	5.58	d		A111	0.53	c		A238	369.75	d
A124	5.47	d		A315	0.53	c		A447	365.57	d
A332	5.40	d		A328	0.52	c		A236	353.53	d
A314	5.40	d		A336	0.51	c		A189	350.41	d
A237	5.28	e		A154	0.51	c		A235	322.71	d
A356	5.09	e		A317	0.50	c		A355	315.43	d
A334	5.08	e		A318	0.50	c		A333	298.04	e
A161	4.98	e		A124	0.48	c		A331	283.75	e
A331	4.93	e		A337	0.48	c		A423	278.33	e
A338	4.89	e		A327	0.48	c		A332	275.86	e
A160	4.89	e		A302	0.48	c		A356	260.79	e
A423	4.72	f		A316	0.48	c		A330	260.32	e
A315	4.71	f		A160	0.48	c		A224	244.97	e
A372	4.52	f		A335	0.47	c		A314	244.94	e
A330	4.48	f		A329	0.46	c		A372	243.74	e
A236	4.47	f		A314	0.46	c		A338	241.59	e
A162	4.44	f		A334	0.46	c		A190	236.20	e
A335	4.34	f		A161	0.45	c		A337	233.06	e
A259	4.32	f		A355	0.45	c		A373	228.86	e
A260	4.27	g		A163	0.41	d		A161	226.83	e
A154	4.26	g		A162	0.41	d		A124	226.63	e
A336	4.19	g		A338	0.41	d		A334	224.43	e
A235	4.14	g		A333	0.40	d		A162	221.23	e
A373	4.13	g		A356	0.39	d		A191	220.29	e
A329	4.08	g		A332	0.39	d		A424	216.02	e
A238	4.08	g		A164	0.38	d		A160	211.42	e
A316	4.05	g		A424	0.37	d		A163	199.53	e
A163	4.04	g		A189	0.37	d		A164	198.27	e
A424	4.04	g		A372	0.37	d		A335	186.70	f
A155	4.02	g		A499	0.37	d		A329	179.16	f
A158	4.00	g		A373	0.36	d		A315	178.97	f
A113	3.98	g		A331	0.35	d		A316	176.06	f
A317	3.97	g		A330	0.35	d		A154	171.16	f
A159	3.92	g		A447	0.35	d		A336	163.84	f
A318	3.89	g		A423	0.34	d		A318	161.85	f
A190	3.87	g		A190	0.33	d		A327	159.33	f
A327	3.81	h		A191	0.33	d		A317	158.71	f
Ni	3.76	h		A224	0.31	d		A159	151.28	f
A126	3.75	h		A226	0.29	e		A155	151.17	f
A156	3.74	h		A225	0.28	e		A328	148.09	f
A328	3.72	h		A235	0.26	e		A113	142.61	f
A224	3.72	h		A236	0.26	e		A126	134.71	f
A164	3.68	h		A237	0.23	f		A111	132.61	f
A258	3.65	h		A238	0.23	f		A158	128.06	f
A191	3.54	h		A257	0.21	f		A156	119.71	f
A157	3.48	h		A259	0.20	f		A115	113.19	f
A111	3.47	h		A258	0.19	f		A125	111.50	f
A115	3.37	h		A260	0.18	f		A157	105.09	f
A125	3.28	h		Ni	—			Ni	—

Means followed by the same letter in the columns do not differ by the Scott-Knott 5% test. Ni: Culture medium not inoculated with fungi and incubated together with the other strains treatments.

Publication Dates

• Publication in this collection
  06 Dec 2024
• Date of issue
  2024

History

• Received
  18 Dec 2023
• Accepted
  9 Sept 2024