Plant growth-promoting activity of wild-type and bromate- resistant mutant of the endophytic fungus Colletotrichum karstii

Endophytes may play important roles in agriculture. Spontaneous or induced mutant strains may increase their biotechnological properties. Seventeen Colletotrichum endophytic fungi were investigated for their plant growth-promoting characteristics (in vitro phosphate solubilization, IAA, and siderophore production). The five best strains were inoculated into bean seeds, and the most prominent isolate was selected to obtain auxotrophic mutants by Potassium Bromate Resistance System (PBRS). The plant growth-promoting ability of the mutant was also investigated. Further, 41.17% of the evaluated endophytes presented promising results for in vitro assays (C. karstii SL10, C. karstii SL28, C. karstii SL57, C. karstii SL59, C. karstii SL12, C. karstii SL40, and C. karstii SL24). The endophyte C. karstii SL57 was statistically conspicuous for plant height and root length when compared to those in control plants. Bromate-resistant mutant C. karstii SL57 increased in vitro phosphate solubilization (23%) and chlorophyll levels (Chlb 0.607 mg g and Chlt 0.973 mg g) of bean plants when compared to the wild-type strain (Chlb 0.551 mg g and Chlt 0.881 mg g). This is the first time an auxotrophic mutant fungus has been obtained by PBRS with a biotechnological application for the agricultural field.


Introduction
In 2017, the global bean production was around 24 million tons planted in one million hectares, with approximately 109 million tons of nitrogen and 45 million tons of phosphate fertilizers (Food and Agriculture Organization of the United Nations [FAO], 2020). Nitrogen and phosphates were required during the planting stage to control plant nutritional deficiencies, since several micronutrients, such as phosphorus, are not easily absorbed by plants (Wang & Wang, 2016).
The Colletotrichum genus is among the most frequently isolated endophytic fungi. Although the genus has already been considered one of the eight pathogens of great agricultural importance (Cannon, Damm, Johnston, & Weir, 2012), the latter have also proved to be able to associate endophytically with different hosts and perform activities of biotechnological importance, such as enzyme production (Santos et al., 2019), phosphate solubilization (Hiruma et al., 2016) and the production of plant hormones (Robinson, Riov, & Sharon, 1998).
In current study, seventeen leaf endophytic fungi belonging to eight Colletotrichum species have been investigated for their plant growth-promoting agent characteristics. The ability to solubilize phosphate to produce IAA and in vitro siderophores of all endophytes was also evaluated. The five best strains were analyzed Toxicity of the Colletotrichum karstii SL57 wild strain to potassium bromate The endophyte C. karstii SL57 with a statistical emphasis for in vitro assays and plant growth-promotion on common bean plants was used to obtain spontaneous auxotrophic mutants resistant to potassium bromate.
Colonies with no growth on MM with bromate were considered as putative auxotrophic mutants. The site where they were inoculated in the MM solid medium was cut and transferred to CM. After 7 days of incubation at 28°C, each isolate was tested for nutritional conditions, as described by Cordeiro, Lima, and Azevedo (1995), in a selective medium containing bromate (25 mM) and one single nitrogen source (5 mM).

Plant growth-promotion capacity of the mutant strain
The in vitro solubilization of inorganic phosphate and IAA assays were tested according to the previously mentioned methodologies for the wild-type strain. Bromate-resistant mutant strain in common bean seeds was also inoculated as previously described. However, plants were incubated for 15 days at 28ºC, with a photoperiod of 8h light/ 16h dark and four replications. In addition to the plant height, root length and number of leaves, all the plants´ fresh root, dry weight and chlorophyll were also determined.
Further, 1 g of fresh matter from each plant treatment was macerated in 10 mL of 80% acetone solution to determine chlorophyll concentration (mg g -1 ). The acetone extract was centrifuged at 3000 xg for 20 min., and the supernatant was analyzed in a spectrophotometer at 645, 652, 654, and 663 nm. Calculations to determine the milligrams of chlorophyll per gram of leaf fresh tissue were based on equations by Whitham, Blaydes, and Devlin (1971): Chlorophyll a (Chla) = (12.7 x A663 -2.69 x A645) V 1000W -1 , Chlorophyll b (Chlb) = (22.9xA654 -4.68 x A663) V 1000W -1 , and total chlorophyll (Chlt) = (A652 x 1000 x V 1000W -1 )/ 34.5; where A = Absorbance at the indicated wavelength; V = final volume of chlorophyll -acetone extract; W = Fresh leaf matter in grams of the plant material used.

Statistical analysis
All tests were performed in triplicate, except the inoculation of endophytes in bean seeds, which were analyzed in four replicates for each treatment. Averages of data underwent ANOVA (analysis of variance) and statistically compared by Scott-Knott test at p < 0.05, employing the SISVAR statistical analysis program (Ferreira, 2011).

Results and discussion
Although the genus Colletotrichum has been commonly described as an agricultural important phytopathogen (Cannon et al., 2012), these fungi have also had their endophyte role successfully reported in different plant species (Bongiorno et al., 2016;Ribeiro et al., 2018;Santos et al., 2019;Golias et al., 2020;Oliveira et al., 2020).
Several studies with endophyte promoting plant-growth have focused on endophytic bacteria (Sánchez-Cruz et al., 2019) and the identification and selection of endophytic fungi have also increased. Two important characteristics for plant growth-promotion by microorganisms are the ability to solubilize inorganic phosphate and to produce plant hormones, such as IAA. Table 1 shows in vitro assays for mechanisms that may indicate growth-promoting agent characteristics. For the quantification of IAA using the commercial hormone curve (R 2 =0.98), significant statistical differences were observed for strains C. gigasporum JB168 (144.32 μg mL -1 ) and C. cacao JB41 (205.87 μg mL -1 ) when compared to remaining strains. Although the endophytes C. truncatum JB277, C. karstii SL57, C. plurivorum JB47, C. karstii SL40, C. phyllanthi SL24, and C. gigasporum JB160 were assembled into a different statistical group, significant amounts higher than 70 μg mL -1 were also reported. Several ascomycete fungi have been capable of producing IAA, a hormone for plant growth and development of paramount importance. In vitro biosynthesis of IAA using tryptophan as a precursor is the most commonly reported type. Robinson et al. (1998) quantified the IAA amount production by 18 strains of Colletotrichum sp., describing results ranging between 2 and 32 μg mL -1 . In current research, Colletotrichum strains which were capable of producing 2 to 144 μg mL -1 amounts of IAA have been described. These rates are higher than those reported in the study by Robinson et al. (1998) and also in other research works such as that by Ye, Li, Yi, Zhang, and Zou (2019), who employed different endophytic fungi, with the best isolate producing about 85 μg mL -1 .
In a medium containing inorganic phosphate (CaHPO 4 ), 52.94% of the endophytic fungi presented a solubilization halo around the fungal colony, suggesting a solubilizing activity. Nine isolates showed a solubilization index ranging between 2.12 and 2.91 cm. According to statistical analysis (Scott-Knott test at 5% significance), the endophyte C. karstii SL60 was conspicuous by presenting a solubilization index of 2.91 cm ( Figure 1). Table 1 shows the production of siderophores expressed in AU (Activity Unit). Twelve strains showed a positive result, with an activity index between 0.08 and 0.65 AU. The activity of solubilizing phosphate performed by endophytes is also important for their hosts, since phosphate is one of the three macronutrients that limit plant growth. However, most phosphorus present in many soils exists in insoluble and unavailable forms for plants (Wang & Wang, 2016). Hiruma et al. (2016) studied the colonization of C. tofieldiae in Arabidopsis thaliana plants and suggested, through Colletotrichum transformants with the GFP gene, that colonization by this genus of fungi begins through the root system and is then systematically distributed to other tissues. Another important result reported by Hiruma et al. (2016) is the ability of C. tofieldiae of transferring phosphate to the treated plants and thus contributing to their development.
In current assay, 41.17% of the evaluated Colletotrichum endophytic fungi presented promising results for IAA production, phosphate solubilization, and siderophores synthesis (C. karstii SL10, C. karstii SL28, C. karstii SL57, C. karstii SL59, C. karstii SL12, C. karstii SL40 and C. karstii SL24). However, it may be underscored that, although endophytes C. gigasporum JB168 and C. cacao JB41 have not presented a phosphate solubilization index and siderophores production, they were still able to synthesize large amounts of IAA in a L-Tryptophansupplemented medium (5 mM) ( Table 1). Table 2 outlines the results obtained for the biometric parameters of inoculated and non-inoculated bean seedlings with the endophytes C. karstii SL57, C. phyllanthi SL24, C. karstii SL40, C. cacao JB41 and C. gigasporum JB168. No visible symptoms of diseases were observed in the plants treated with the suspension of endophytes and their respective control plants, suggesting an endophytic association. Significant statistical results were reported on the number of leaves for treatments with C. phyllanthi SL24 (5.0) and C. karstii SL40 (5.0) when compared to control plants (3.5). There was also a statistical difference in shoot height for treatments C. karstii SL57 (20.87 cm), C. phyllanthi SL24 (18.25 cm), C. karstii SL40 (19.85 cm) and C. cacao JB41 (21.12 cm) in relation to the untreated control plants (15.35 cm) (Figure 2).
When length of roots ( Figure 2) was assessed, C. karstii SL57 (21.0 cm) was outstanding among the other endophytes' treatments and control plants (16.50 cm). In fact, three endophytes (C. phyllanthi SL24, C. karstii SL40 and C. karstii SL57) stood out among the biometric parameters analyzed. The above indicates their potential as growth-promoting agents, especially in bean plants (Table 2). When the positive roles presented by endophytic microorganisms for their hosts are taken into account, genetic improvement using endophytic fungi or bacteria, such as the isolation of spontaneous or induced mutants, may lead to the discovery of new strains with the improvement or introduction of new biotechnological characteristics of importance for their hosts (Freeman & Rodrigues, 1993;Prusky, Freeman, Rodriguez, & Keen, 1994;Pamphile, Rocha, & Azevedo, 2004).
Since endophyte C. karstii SL57 has shown significant statistical results for in vitro assays (Table 1) and has stood out in two biometric parameters when compared to the bean control plants and other endophytes treatments (Table 2), the strain was selected for isolating auxotrophic mutants resistant to potassium bromate aiming at obtaining mutant strains capable of maximizing their activities as a plant growth-promoting agent.
The toxicity of potassium bromate to the wild strain of C. karstii SL57 was achieved at a concentration of 25 mM, with complete inhibition occurring at 50 mM ( Table 3). The concentration of 25 mM was chosen for isolating the bromate resistant mutants considering the toxicity of 87%, since 50 and 100 mM were very restrictive (100% toxicity). The evaluation was carried out in Complete Agar Medium supplemented with different concentrations of Bromate. All concentrations were tested in triplicate and incubated at 28ºC for 7 days.*Average growth in triplicates. Table 4 demonstrate the putative mutants of C. karstii SL57 strain resistant to the concentration of 25 mM of potassium bromate. Although 40 putative mutants were screened for five Nsources (Cys, Met, Lys, UA, and Cyst), only one mutant was confirmed for uric acid (C. karstii SL57 mut BR-UA ). A 6 mm-diameter plug of the putative mutant colonies were grown on Minimum medium + 25 mM bromate + each N-source. Those fungi that failed to grow on MM supplemented with bromate + N-source but failed on unsupplemented MM were considered an auxotrophic mutant. All plates were incubated at 28ºC.

Results presented in
The description of auxotrophic mutants of the Colletotrichum genus has been previously described, especially strains resistant to potassium chlorate (KClO 3 ) (Prado et al., 2007;Rosada et al., 2010;Santanna et al., 2010). In 2011, Carvalho and Costa (2011) studied the genetic variability and vegetative compatibility of nit mutant strains of C. lindemuthianum and reported, similar to the authors of current study, auxotrophic mutants for uric acid (UA). Most studies aimed at isolating Colletotrichum mutants use the chlorate resistance system, while in current assay the authors are reporting for the first time retrieval of Colletotrichum mutants through the potassium bromate resistance system (KBrO 3 ), similar to the one previously mentioned for the Aspergillus species (Kanan, 2002;Kanan & Al-Najjar, 2010).
Significant statistical results were observed according to Scott-Knott test (5% significance) for the phosphate solubilizing activity of C. karstii SL5 mutant strain when compared to the wild-type strain. Results in Table 5 demonstrate a 23% increase in the solubilization index by the mutant fungus. In the case of IAA production ( Figure 3B), there was a decrease in the ability to synthesize this hormone in vitro by the mutant strain. However, the mutant strain continued to produce a significant amount of auxin when compared to other Colletotrichum wild-type strains described in Table 1, with similar indices.  When inoculated in common bean seeds ( Figure 3A), C. karstii SL57 mut BR-UA was able to demonstrated the permanence of their plant growth-promoting characteristics. Although no statistical differences were observed between the wild-type and mutant strain for the biometric parameters analyzed, both C. karstii SL57 wild and C. karstii SL57 mut BR-UA statistically stood out among the control plants (Table 6 and 7), especially for the shoot fresh weight and root fresh/dry weight (Table 8).  We have inoculated the wild-type strain and Colletotrichum mutant by superficially disinfected seeds. According to findings by Hiruma et al. (2016), these strains may have initiated colonization by their roots, or rather, one of the first stages of bean development, suggesting that the statistical differences found in the root lengths of the plants treated with C. karstii SL57 wild strain (21 cm) after 21 days may be related to this colonization mechanism. Further, the phosphate solubilization index of wild C. karstii SL57 (2.53 cm) increased in C. karstii SL57 mut BR-UA (2.76 cm), which would reinforce the fresh and dry biomass of the plants analyzed after 15 days when compared to control plants (0.65 g), may also be related to the authors' findings.
Chlorophyll contents demonstrated the best results. The C. karstii SL57 mut BR-UA strain were statistically prominent both for chlorophyll b (0.6078 mg g -1 ) and total chlorophyll (0.9734 mg g -1 ) when compared to control plants and plants treated with wild-type endophyte (Table 8, Figure 3C). Chlorophyll is a molecule of extreme importance for plants. It is related to photosynthesis and is responsible for capturing and transmitting light energy during the first stage of the photosynthetic process (Song et al., 2020). The most common types of chlorophyll found in plants are type Chla and Chlb (Costa et al., 2019).
The binding of Chlb with antenna proteins is crucial for the correct assembly of antenna complexes on thylakoid membranes. Therefore, Chlb levels affect the capture of light and thermal energy dissipation processes and the electron transport in the thylakoid membrane (Hoober, Eggink, & Chen, 2007;Voitsekhovskaja & Tyutereva, 2015). Further, Chlb is related to delay of cell aging and photosynthetic processes, making them longer. The latter are characteristics previously reported in transgenic and non-transgenic plants for the production of this type of chlorophyll (Tyutereva & Voitsekhovskaja, 2011;Biswal et al., 2012).
Longer photosynthesis periods could lead to higher biomass accumulation in plants, which is a particularly interesting characteristic for agriculture, since it would allow an increase in productivity (Voitsekhovskaja & Tyutereva, 2015). These ideas corroborate our findings with the C. karstii wild-type and mutant strains inoculated and analyzed at 15 days in common bean plants. The Chlb levels in plants treated with C. karstii SL57 mut BR-UA suggest that the strain may have provided longer photosynthetic periods to plants and favored the accumulation of biomass, which statistically differed from that in control plants.
In current assay, the isolation of a bromate-resistant auxotrophic mutant with a single nutritional requirement has been reported. Detected mutations, probably in one single locus to acid uric metabolism, were capable of producing advantageous mutations. This may be observed in a study by Freeman and Rodriguez (1993) with C. magna mutant resistant to potassium chlorate, which reduced the virulence and pathogenicity in watermelon when compared to its wild-type strain, without losing important characteristics such as sporulation and the ability to infection/colonization. Prusky et al. (1994) corroborate results by Freeman and Rodriguez (1993) regarding the positive mutation in C. magna chlorate-resistant strains, now in avocado plants. The authors' findings corroborate results in our study for Colletotrichum mutants, especially the C. karstii SL57 mut BR-UA auxotrophic mutant, which presented an increase in solubilization activity of inorganic phosphate (23%) and the Chlb and Chlt contents in common bean plants. López-González et al. (2017) and Oliveira et al. (2020) have reported the Colletotrichum fungi playing an endophyte role in plants of Phaseolus sp. genus, similar to reports by Parsa et al. (2016), including the C. karstii species analyzed in current study and described as a bean endophyte by the authors. Thus, the above authors' findings and results of current assay foreground the hypothesis that the genus of fungi may associate itself endophytically and act as plant growth-promoting agent, especially in bean plants.

Conclusion
Current assay showed that endophytic fungi of the Colletotrichum sp. genus were able to solubilize inorganic phosphate, producing IAA/siderophores, and promoting bean growth. Further, we have described for the first time an auxotrophic mutant strain for isolated uric acid using the potassium bromate resistance system, with a biotechnological application for the agricultural field. The mutant C. karstii SL57 mut BR-UA in vitro increased the phosphate solubilizing capacity and the chlorophyll levels of bean plants when compared to the wild-type strain.
Strains of spontaneous or induced mutant microorganisms, especially the endophytes, are important because these microorganisms will actually use their hosts' nutritional sources to supply their auxotrophic needs. If released into the environment, they will not cause any harmful effects.