FUNGI ASSOCIATED WITH THE RHIZOSPHERE OF Rhizophora mangle AND THEIR RELATIONSHIP WITH THE NATURAL ATTENUATION OF PETROLEUM-CONTAMINATED SOILS †

The strains were identified by cell morphology and rDNA ITS region sequencing, and the sequences were used to construct a phylogenetic tree using the maximum likelihood method. A liquid culture was developed in a mineral medium added with petroleum for 30 days and the saturated, polar, and aromatic fractions were quantified to determine the percentage of biodegradation. Results. Six strains of hydrocarbonoclastic fungi were isolated and identified in R. mangle rhizosphere contaminated with 109 916.5 mg kg -1 of TPHs. Of these strains, Aspergillus niger and A. flavus showed the highest hydrocarbon biodegradation with 30.5 and 26.1%, respectively. The highest biodegradation of the saturated fraction was observed with A. niger , A. flavus , and A. egyptiacus ; Fusarium oxysporum and A. niger preferred the polar fraction, while A. niger and A. flavus assimilated more of the aromatic fraction. Implications. These hydrocarbonoclastic strains may be potentially used in restoration strategies for hydrocarbon-contaminated mangroves. Conclusion. The microorganisms associated with contaminated mangroves are part of the natural attenuation of the studied site and may be useful for the treatment of sites affected by petroleum spills.


INTRODUCTION
The coverage of mangrove ecosystems has suffered a decline worldwide mainly due to anthropogenic factors.In Mexico, important mangrove areas are found in the North, Central, and South Pacific, the Yucatán Peninsula, and the Gulf of Mexico (Rodríguez-Zuñiga et al., 2013).In the latter, the littoral zone of the state of Veracruz represents 4.96% of the mangrove-covered surface area of Mexico (Cuevas-Díaz et al., 2020).In the south of Veracruz, mangroves such as Avicennia germinans, Laguncularia racemosa, Rhizophora mangle, and Conocarpus erectus have been affected by petroleum contamination, mainly along the Coatzacoalcos River, due to spills caused by accidents in oil wells and storage tanks, as well as poor management, lack of maintenance of pipelines, and criminal activity.Mangrove areas in the Coatzacoalcos River are affected by oil spills because they are sites surrounded by large industrial complexes that belong to Petróleos Mexicanos (PEMEX) and private companies.Petroleum contamination in mangrove ecosystems is a serious environmental problem because there is a constant loss of diversity and ecosystem services are thus affected.There are native microorganisms that participate in the natural restoration of ecosystems, such as bacteria and fungi (Kumar andGopal, 2015, Koshlaf andBall, 2017).Some genera of fungi can grow in the presence of hydrocarbons (Chaurasia et al., 2019, Al-Hawash et al., 2018), the hydrocarbonoclastic activity allows these fungi to biotransform different hydrocarbon fractions.Some hydrocarbon biodegradation studies have used basidiomycetes such as Chrysosporium, Phanerochaete, Pleurotus ostreatus and Trametes versicolor (Yateem et al., 1998, Mollea et al., 2005).Similarly, filamentous fungi are used to treat petroleum-contaminated soils through remediation processes (Benguenab and Chibani, 2021).This bioremediation potential is mainly due to the characteristics of the enzyme systems and the growth of these fungi, which enables them to develop mycelia, colonize different types of substrates, and access toxic compounds (D'Annibale et al., 2006).The high cell surface area to cell volume ratio of filamentous fungi makes them efficient degraders in certain niches such as soil contaminated with toxic organic molecules (Martín et al., 2004).In this context, there are reports of fungi such as Alternaria alternata, Aspergillus flavus, Curvularia lunata, Fusarium solani, Mucor racemosum, Penicillium notatum and Ulocladium atrum that have been isolated from petroleumcontaminated soil in Saudi Arabia (Hashem, 2007).Furthermore, Mohsenzadeh et al. (2010) reported fungal species associated with the roots of Polygonum aviculare located in a petroleum-contaminated area in Iran, which included Alternaria, Aspergillus, Bipolaris, Fusarium, and Rhizoctonia.Considering the above, the objective of the present study was to isolate fungal strains associated with the rhizosphere of Rhizophora mangle contaminated with hydrocarbons and determine their hydrocarbonoclastic potential in culture media added with petroleum.

Study area and sampling
Five 0.5-kg samples of the rhizosphere of Rhizophora mangle, known as red mangrove, were collected at a depth of 10 cm in a plot of 100 m 2 (Yang et al., 2021;Mahmoudi et al., 2022) in the locality of El Polvorín (Figure 1), located in the municipality of Cosoleacaque, Veracruz (18°3'42.42ˮN,-94.25'7.91ˮO).This area is affected by contamination from spills of Maya crude oil (API 21.85) caused by constant illegal tapping from Nuevo Teapa to Poza Rica, Veracruz, Mexico.The 0.5-kg soil samples were collected in three sterile containers in a systematized section of R. mangle monitored in a zigzag fashion.The samples were then dried and sieved with a mesh of size 2 and kept at 4 °C for subsequent physicochemical and microbiological analyses (Paez and Wilke, 2005).

Physicochemical characterization of the soil
A composite sample was made from the samples obtained using the shaking extraction method following the US EPA 3500B and US EPA 3540C (1996) methods and Schwab et al. (1999), with some modifications in shaking speed and solvent volume (Arce-Ortega et al., 2004).Extraction was performed with 2 g of sample previously dried at 25 °C for 48 h, which were subsequently added with 3 g of anhydrous Na2SO4 and mixed in 15-mL tubes with a vortex mixer.The mix was then added with 5 mL of CH2Cl2, shaken for 45 s, and then centrifuged at 6,000 rpm for 10 min.The supernatant was removed and placed in a roundbottom flask.This process was repeated three times.Finally, the CH2Cl2 was evaporated to dryness in a rotary evaporator under reduced pressure.The results are expressed in mg of TPH/kg of dry soil (total petroleum hydrocarbons, mg/kg of d.s.), which was calculated as follows: TPH=(RB-RA)*(CF)/(P*HF). Where: RA= weight of the empty container at constant weight (mg); RB= weight of the container with the concentrated organic extract (mg); P= quantity of extracted soil (g); HF= humidity correction factor (1 -(% humidity/100)); CF= correction factor to transform to kg of d.s.= 1000.

Isolation of hydrocarbonoclastic fungal strains
The strains were isolated by the plate dilution method (1x10 -4 ) from 10 g of sample.A 1-mL aliquot from each dilution was placed on Petri plates, which were subsequently added with mineral agar (g/L): Noble agar, 15; KH2PO4, 0.4; K2HPO4, 1.6; NH4Cl, 1.5; MgCl2•6H20, 0.17; Na2SO4, 0.609, and CaCl2•2H2O, 0.045; acidified with lactic acid at a pH of 4.9 and placed on the lid of the Petri plate with paper impregnated with petroleum previously sterilized three times at 121 °C and 15 psi of pressure for one hour.Finally, they were incubated for 7 days at 25 ± 2 °C.The criteria for the selection and purification of the hydrocarbonoclastic fungal strains were their tolerance and capacity to develop in a microenvironment saturated with volatile petroleum hydrocarbons (Fernández et al., 2006).The strains that developed mycelia in the presence of volatile hydrocarbons were then cultured in cellulose agar (g/L): crystalline cellulose, 5; (NH₄)₂SO₄, 5; KH2PO4, 1; MgSO4•7H2O, 5; rose bengal (0.001); yeast extract (0.025), and bacteriological agar, 20.A 1.5 cm 2 -piece of filter paper impregnated with Maya crude oil was placed on each Petri plate containing cellulose agar.A hole punch was used to obtain one slice of 1 cm in diameter of each purified fungal strain, which was placed on the filter paper impregnated with Maya crude oil in the cellulose agar medium.Finally, they were incubated at 25 ± 2 °C for 7 days (Rivera et al., 2004).

Identification of hydrocarbonoclastic strains
The fungal strains that showed the capacity to utilize hydrocarbons as a carbon source were identified by their colony morphology and microscopic characteristics (Barnett and Hunter, 1998), and a molecular analysis was performed to corroborate the identity of the species.This was done by isolating the DNA following Yu et al. (2011), with some modifications.The rDNA ITS region was subsequently amplified using the primers ITS1 and ITS4.The amplified products were purified using the Wizard kit (Pro-mega) and nucleotides were sequenced in the Biotechnology Institute of UNAM using a sequencer (Applied Biosystems).Finally, the nucleotide sequences obtained were edited in BioEdit version 7.0.5.3 and compared with those in NCBI's GenBank (http://www.ncbi.nlm.nih.gov/) using BLAST to confirm the identity of the species.

Biodegradation capacity of the hydrocarbonoclastic strains
The biodegradation percentage (%B) of each hydrocarbonoclastic fungal strain was obtained following the methodology by Outdot et al. (1987).For this, a liquid culture was prepared in a mineral medium (MM) with the following composition (g/L): KCl, 0.25; NaH2PO4, 1; MgSO4, 0.5; NO3NH4, 1; added with 0.1 g/L of chloramphenicol and with a pH adjusted to 6.2.We used 250 mL Erlenmeyer flasks with 70 mL of MM, 100 μL of sterile Maya crude oil, and 1x10 6 conidia/mL.The abiotic control consisted of MM and Maya crude oil.They were subsequently incubated at 25 ± 2 °C for 30 days.After this time, each flask was extracted three times with 30 mL of CH2Cl2 in separatory funnels and the extracts were placed in 250-mL round-bottom flasks and subsequently evaporated to dryness in a rotary vacuum evaporator to continue monitoring each round-bottom flask at constant weight.All the assays were performed in triplicate.The biodegradation percentage (%B) of each strain was determined according to the following formula:

%B= 100 (MC-M)/MC
Where: MC= extract mass in the control; M= mass in the culture.The extract obtained from the determination of the biodegradation (%B) of each fungal strain was used to determine the percentages of the saturated, aromatic, and polar hydrocarbon fractions by column chromatography using 100 mesh silica gel as the stationary phase and successive elutions of 60 mL of hexane, benzene, and methanol, respectively, as the mobile phase in chromatographic columns of 300 x 15 mm (Chaîneau et al., 1995).

RESULTS AND DISCUSSION
The physicochemical parameters of the composite rhizosphere sample are shown in Table 1.The soil was characterized as a silt loam soil with good water retention capacity, medium natural fertility, and a mildly acidic to neutral pH.It contained 109 916 mg kg -1 of TPHs, which exceeds the maximum permissible limit (MPL) established by the standard NOM-138-SEMARNAT/SS-2012, which mentions that industrial soil can contain up to 6 000 mg kg -1 of heavy fractions and 5 000 mg kg -1 of medium fractions.Mangroves are known to show high productivity due to their abundant organic detritus, which allows the accumulation of hydrocarbons (Olguín et al., 2007).
According to Dragun and Barkack (2000), the recommended pH range for aerobic hydrocarbon degradation in soil is between 5 and 9, with an optimal value of 7. The content of organic nitrogen is below the required value for the composition of microbial cells, which is 14% according to Vidali (2001); however, hydrocarbon bioremediation processes show better results with C:N ratios of 100:10 to up to 100:1 (Van Hamme et al., 2003).The C/N ratio (3.9/0.3)observed in our soil was 11.8, which is consistent with the C/N ratio of 5-17 required for hydrocarbon biodegradation in soil by microscopic fungi (Sterner and Elser, 2002).
Five morphologically different strains of microscopic fungi were isolated and purified, which were able to develop in and tolerate a microenvironment saturated with volatile petroleum hydrocarbons, as well as to reproduce while in direct contact with Maya crude oil (Figure 2).The hydrocarbonoclastic strains were characterized based on their colony morphology and microscopic characteristics, where four of them (H3S2, H4S1, H4S5, and H3S3) showed similarity to the genus Aspergillus.The species A. flavus showed radial to columnar conidial heads, a generally hyaline conidiophore, and metulae that cover three fourths of the vesicle.
Aspergillus terreus shows light brown conidial heads that form compact columns, spherical or pyriform vesicles, and globose or ellipsoidal conidia.
Aspergillus niger is characterized by radiate conidial heads, conidiophores with a thick wall and a brown apex, spherical vesicles, metulae that cover the whole surface of the vesicle, and phialides with generally rough globose conidia.Aspergillus egyptiacus shows abundant conidial structures and conidiophores that are mostly not arranged in a typical Aspergillus head but in solitary phialides or small groups.Finally, the strain H3S6 is consistent with the description of Fusarium oxysporum, which has crescent-shaped, hyaline, septate macroconidia, as well as oval microconidia (Barnett and Hunter, 1998;Geiser et al., 2007).
The morphological identification of the fungal isolates with hydrocarbonoclastic capacity was corroborated by sequencing the rDNA ITS region, whereby the amplification products of each fungal isolate were obtained at 600 bp.The following results were obtained from the nucleotide sequence analysis and their comparison in GenBank: the isolate H4S5 showed 100% similarity with and was grouped into the clade of Aspergillus niger Tiegh, the isolate H3S3 showed 99% similarity with and was grouped into the clade of Aspergillus egyptiacus Moub.& Moustafa, and the isolate H3S6 showed 100% similarity with and was grouped into the clade of Fusarium oxysporum Schltdl.The isolates H3S2 and H4S1 showed 99% similarity with Aspergillus flavus Link and Aspergillus terreus Thom, but the results of the cladogram were inconclusive and thus it was not possible to group them with these species (Figure 3).
These results agree with what has been reported by different authors, where the genera of hydrocarbonoclastic filamentous fungi that predominate in these sites are Aspergillus, Penicillium, and Fusarium.Strains of Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Emericella nidulans, Fusarium solani, Penicillium funiculosum, Rhizopus stolonifer, and Trichoderma harzianum have been isolated from kerosene-and benzene-contaminated soil in Egypt (Hemida et al., 1993).Colombo et al. (1996) isolated Aspergillus terreus and Fusarium solani from hydrocarbon-contaminated soil in Argentina and April et al. (2000) isolated 64 species of filamentous fungi from soil contaminated with crude oil in Canada, where only 6 genera showed the capacity to degrade hydrocarbons.Furthermore, Hashem (2007) reported the isolation of Alternaria alternata, Aspergillus flavus, Curvularia lunata, Fusarium solani, Mucor racemosum, Penicillium notatum, and Ulocladium atrum from petroleum-contaminated soil in Saudi Arabia.Similarly, Mohsenzadeh et al. (2010) reported fungal species associated with the roots of Polygonum aviculare in a petroleum-contaminated area in Iran, which included Alternaria, Aspergillus, Bipolaris, Fusarium, and Rhizoctonia.There is also a review of the isolation of fungi such as Penicillium, Aspergillus, Fusarium, and Rhizopus from hydrocarbon-contaminated soil and water (Pernía et al., 2012).However, even though many fungal species have been isolated from this type of site, not all can degrade hydrocarbons.
The total petroleum hydrocarbon biodegradation observed is shown in Figure 4, where the strain H4S5 of A. niger showed the highest biodegradation percentage (30.5 %).
Once the biodegradation percentage of each fungal isolate was determined, the TPH composition (saturated, aromatic, and polar) was estimated to evaluate the capacity to biodegrade each TPH fraction (Figure 5).The results showed that the saturated fraction had the highest biodegradation percentages by all the fungal strains studied, followed by the polar fraction, and, to a lesser degree, the aromatic fraction.The strains that showed the highest consumption of the saturated fraction were A. niger, A. flavus, and A. egyptiacus, while F. oxysporum and A. niger consumed a higher percentage of the polar fraction than the other strains, and A. niger and A. flavus consumed a higher percentage of the aromatic fraction.The genera Penicillium, Aspergillus, and Fusarium are distinguished by their hydrocarbonoclastic capacity (Naranjo et al. 2007;Chaîneau et al., 1999;Pernía et al., 2012).The genus Aspergillus has been particularly widely described because of its capacity to metabolize TPHs (Oudot et al., 1993;Chaîneau et al., 1999;Benguenab and Chibani, 2021).The TPH biodegradation capacity of these strains showed values from 15.2 to 30.5%, which agrees with the results reported by Chaîneau et al. (1995) for strains of the genera Penicillium, Aspergillus, and Fusarium.The genus Aspergillus has been reported to grow rapidly in crude oil and, in some cases, can form very dense hyphal networks (April et al., 2000).Moreover, Oudot et al. (1993) reported that this genus can degrade between 30 and 35% of saturated and aromatic hydrocarbons and 13% of resins and asphaltenes, which are constituents of crude oil.Saturated hydrocarbons showed the highest biodegradation percentages, which agrees with that observed by Chaîneau et al. (1999), who reported a higher degradation of this hydrocarbon fraction and demonstrated that the linear chains and alkane range were partially degraded by Aspergillus niger, Penicillium resctrictum, Brevandimonas vesicularis, and Trichoderma harzianum.The filamentous fungi identified in the present study showed an adaptive capacity to develop and metabolize TPHs as a carbon and energy source in plates with cellulose agar and petroleum, where mycelial growth was observed from the third day of incubation.

CONCLUSIONS
The native fungal isolates associated with the rhizosphere of Rhizophora mangle with the highest potential belong to the genera Aspergillus and Fusarium, since they were able to utilize hydrocarbons as a sole source of carbon and energy after 30 days in a liquid culture in mineral medium.These identified strains may be used in bioremediation or natural attenuation processes in contaminated sites, allowing the restoration of mangroves, which are of great importance due to the ecosystem services they provide to the communities that live in this type of ecosystem.

Figure 1 .
Figure 1.Sampling site where the mangrove affected by hydrocarbon spills is located.

Figure 3 .
Figure 3. Maximum likelihood phylogenetic tree based on ITS sequences of hydrocarbonoclastic fungi from the rhizosphere of R. mangle.Numbers in the nodes are support values using 1000 replicates.
Hernández-Acosta  et al. (2003)  isolated Trichoderma sp., Aspergillus sp., and Mucor sp. from the rhizosphere of Chamaecrista nictitans and Panicum sp. from soils in Minatitlán, Veracruz.These microorganisms were able to degrade crude oil in contaminated soil and the rhizosphere of plants such as maize and beans when inoculated in soil contaminated with crude oil.

Table 1 .
Physicochemical characteristics of the composite sample of R. mangle rhizosphere.