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Boletín de la Sociedad Argentina de Botánica

versión On-line ISSN 1851-2372

Bol. Soc. Argent. Bot. vol.53 no.2 Córdoba jun. 2018

 

Micología - Mycology

Microdochium Bolleyi (Ascomycota: Xylariales): Physiological characterization and structural features of its association with wheat

CAROLINA ROTHEN1*, VICTORIA MIRANDA1, SEBASTIÁN FRACCHIA1, ALICIA GODEAS2 and ALEJANDRA RODRÍGUEZ2

1  Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja (CRILAR), Provincia de La Rioja, UNLAR, SEGEMAR, UNCa, CONICET. Entre Ríos y Mendoza, 5301 Anillaco, La Rioja, Argentina
2 Laboratorio de Microbiologia del Suelo. Instituto de Biodiversidad y Biología Experimental y Aplicada (IBBEA). CONICET-Universidad de Buenos Aires, Intendente Güiraldes 2160. Ciudad Universitaria C1428EGA Buenos Aires. Argentina

*Corresponding author: carorothen@gmail.com


Summary: Plant roots can be colonized by asymptomatic fungal strains belonging to several taxa, among them, the group defined as Dark Septate Endophytes (DSE). Microdochium bolleyi commonly colonizes wheat roots and other crops. It is considered a weak pathogen or even a non-pathogenic fungal species, which has also been considered as a potential biocontrol agent against aggressive soil-borne pathogens in cereal crops. We isolated a strain of M. bolleyi from wheat roots sampled in a crop feld in Argentina, and characterized its abilities to grow in different carbon and nitrogen sources, to produce indole and to solubilize phosphorus; also several enzymatic activities were evaluated. In addition, resynthesis was performed under controlled conditions in order to characterize root fungal colonization under both, optical and transmission microscopy. The strain 22-1 colonized wheat root parenchymal tissue, forming chlamysdospores inside parenchymal cells and root hairs, and poorly grew in carbon and nitrogen sources. This fungus also synthesized indoles in in vitro culture, but it cannot solubilize phosphorus. Only amylase activity was detected out of seven enzymatic activity measured. Microdochium bolleyi (strain 22-1) colonized the roots, it formed typical DSE fungal structures and behaved like a "true endophyte"; however further studies are necessary to elucidate its role in the association with wheat.

Key words: Dark septate endophytes, Microdochium bolleyi, root endophytes, wheat crop.

Resumen: Microdochium bolleyi (Ascomycota: Xylariales): Caracterización fsiológica y caracteres estructurales de su asociación con trigo. Las raíces de las plantas hospedan una gran diversidad de hongos, entre ellos, se encuentran los Endoftos Septados Oscuros (ESO). Microdochium bolleyi coloniza las raíces de trigo y otros cereales, aunque algunos autores lo han considerado un patógeno débil, otros han demostrado su acción biocontroladora contra patógenos agresivos del suelo. En el presente trabajo, se aisló una cepa de M. bolleyi (22-1) de raíces de trigo. Esta cepa fue metabólicamente caracterizada y se realizó un ensayo de resíntesis bajo condiciones controladas con el fin de caracterizar la colonización del hongo en la raíz bajo microscopía óptica y de transmisión. Su crecimiento fue escaso en las fuentes de carbono y nitrógeno evaluadas, sintetizó indoles en cultivo in vitro, pero no mostró habilidades para solubilizar el fósforo, por último, solo se detectó actividad amilasa. La cepa 22-1 coloniza la corteza radicular del trigo, formando clamidosporas melanizadas inter e intracelularmente y en el interior de los pelos radiculares. Microdochium bolleyi (cepa 22-1) coloniza la raíz de trigo formando las típicas estructuras de los ESO y comportándose como un "verdadero endófto", sin embargo, se necesitan más estudios para terminar de dilucidar su papel en la asociación con el trigo.

Palabras clave: Endoftos septados oscuros, hongos endoftos, Microdochium bolleyi, trigo.


 

Introduction

Most plant species can associate with a wide diversity of root endophytic fungi, including the denominated Dark Septate Endophytes (DSE). These endophytes comprise a group of cosmopolitan fungi, mostly of ascomycetes species included in different orders as Helotiales, Pleosporales, Sordariales and Xylariales (Jumpponen, 2001; Jumpponen & Trappe, 1998). DSE are characterized by melanized septate hyphae and microesclerotia or chlamydospores that can colonize the parenchymal tissue of the roots either intra- or intercellularly (Addy et al., 2005; Muthukumar & Tamilselvi, 2010; Sieber & Grünig, 2013), without causing disease symptoms on the host plant (Jumpponen, 2001). Among DSEs, Phialocephala fortinii is one of the best studied taxa, recorded in different environments and plant species (Brenn et al., 2008; Jumpponen & Trappe, 1998). Isolates of this fungus inoculated in several experimental trials, using a range of host plant species and culture conditions (Fernández & Cagigal, 2017; Newsham, 2011; Wilcox & Wang, 1987), yielded positive, neutral or negative effects on plant growth. Similarly, it has been demonstrated that some asymptomatic strains of well-known dark pathogenic fungal taxa (viz. Curvularia spp, Phoma spp.) could be included within this group, as they colonize the root parenchymal tissue with melanized mycelium, behaving as typical DSE fungi (Loro et al., 2012; Priyadharsini & Muthukumar, 2017).

Microdochium bolleyi (syn: Idriella bolleyi) is another well-known species of DSE. It has been extensively isolated from roots and stem bases of several cereals and grasses (Domsch et al., 1980; Salt, 1977). Although it has been reported to cause minor damage under particular conditions, it is mainly considered as non-pathogenic (Kirk & Deacon, 1987; Punithalingam et al., 1979), even M. bolleyi has been patented as a take-all biocontrol agent in cereals and grasses (Fox-Roberts & Deacon, 1988). Mandyam et al. (2010) characterized strains of dark septate endophytes identifed as Microdochium sp. isolated from sampled roots of C4 grasses in Kansas (USA). These authors have demonstrated that two Microdochium sp. isolates used a variety of complex nutrient sources and produced different extracellular enzymes, suggesting the potential role of these fungi in organic matter mineralization and plant nutrition.

Although, DSE studies have focused on descriptive analyses of the colonization and fungal strains, mostly isolated from natural ecosystems (Jumpponen & Trappe, 1998; Newsham, 2011), while, when considering crop species, the information related to their association with DSEs is scarce and limited (Wang et al., 2016; Yuan et al., 2010; Rothen et al., 2017; Fernandes et al., 2015; Muthukumar & Tamilselvi, 2010). Argentina produces approximately 2% of the global wheat production, with an average planting area of 6 million hectares in the last 5 years (Andrade & Satorre, 2015), thus becoming one of the most important crops in the country.

Despite M. bolleyi is commonly found associated with cereals and grasses, forming typical DSE structures, few studies have focused on its physiological abilities and none of them have considered its interaction with wheat from an ultra-anatomical point of view. The aims of this work were: i) to isolate and identify a strain of M. bolleyi from wheat roots and to evaluate physiologically this endophytic fungus and ii) to describe the anatomy of the interaction at two levels, optical and ultra-anatomical, based on resynthesis tests 30 days after inoculation.

MAteriAls And Methods

Isolation and culture

The fungal strain was isolated from wheat roots, sampled in a productive feld in the location of Ferré (Buenos Aires Province, Argentina 34° 06' S-61° 09' W). This area, known as rolling or central sub-humid Pampas, is one of the most productive agricultural regions in the country. The soil is Argiudoll type (pH 7), the annual rainfall, near 900 mm, is concentrated during spring and summer periods and the mean annual temperature is 16°C. Isolation and culture of endophytic fungi from roots were carried out following the methodology described by Silvani et al. (2008). Whole wheat plants (n=15) were sampled in June 2013 and transported to the laboratory within 24hs. The roots were excised and washed prior surface sterilization with 3% NaOCl (v/v) and antibiotics (0.05% w/v Penicillin, 0.05% w/v Ampicillin, 0.05% w/v Streptomycin, 0.05% w/v Tetracycline). The roots were washed with sterile water and cut into segments of 2-3 mm. Then, each segment was transferred to drops of water-agar medium (Gel- Gro®) and incubated at 25°C in the dark. Emerging hyphae from root ends were checked periodically under a binocular microscope and carefully plated onto malt extract agar (MEA) for further growth and fungal characterization. From 24 fungal isolates, one strain (22-1) corresponding morphologically to the genus Microdochium was selected due to observable typical DSE structures. The selection was done through preliminary resynthesis assay with wheat plants.

Morphological and molecular identification

The isolate of Microdochium morphotype (strain 22-1) was cultured on MEA medium to characterize vegetative growth and sporulation. In order to approach the species level identification of the strain, descriptions provided by Ellis (1971) and Hernández-Restrepo et al. (2016) were considered. For molecular identification, isolate 22-1 was cultured in liquid malt extract 20% (w/v) at 25°C for one week. Genomic DNA was extracted using UltraClean™ Microbial Isolation Kit (MO BIO). The internally transcribed spacer (ITS) region of the fungal rDNA was amplified using the primers ITS1 and ITS4. PCR was performed using iProofTM High Fidelity DNA Polymerase (BIO RAD) in a 50 μl reaction volume containing 5–10 ng DNA under conditions described by Triebel et al. (2005). The PCR product was purified with UltraCleanTM PCR Clean-up DNA Purification Kit (MO BIO) and sequenced with primers ITS1 and ITS4 on genotyping service provide by Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, (FCEN-UBA). Sequences obtained from pair primers were aligned to obtain a consensus sequence using Bioedit (Hall, 1999) and compared with others present in the ‘nr’ database with Blastn (Altschul et al., 1990). All sequences were aligned with MAFFT program version 6 (Katoh& Toh 208) available online ( //mafft.cbrc.jp/ alignment/ server/), assigning a cost to the opening 15 and 6 to the extent of the gaps. To infer the phylgenetic tree, the alignments were subjected to a neighbor-joining analysis using the heuristic search option in MEGA v. 5 (Tamura et al., 2013). The support nodes obtained were calculated by the Bootstrap analysis with 1000 replicates (Felsenstein, 1985).

Indole production

For indole production assessment, the fungal isolate was grown in three replicates 250-ml flasks with broth containing (L-1): glucose 2.5 g; sodium succinate 2.5 g; K2HPO4 6 g; KH2PO4 4 g; KOH 2.1 g; NH4Cl 1 g; MgSO4·7H2O 0.2 g; NaCl 0.1 g; CaCl2·2H2O 0.02 g; FeCl3 0.01 g; and Na2MoO4·2H2O 0.002 g (Fuentes-Ramirez et al. ,1993). Liquid medium was supplemented with tryptophan (100μg/L), and incubated at 25°C on a rotary shaker in darkness for 7 days. To detect the presence of indole, 1 mL of the medium was centrifuged (10,000 rpm, 10 minutes) and the supernatant was mixed with an equal volume of the Salkowski chromogenic reagent and incubated for 30 min (Ehmann, 1977). The test was considered positive when a pink-red color change of the supernatant was observed.

Phosphorous biosolubilization

To test the ability of M. bolleyi for phosphorous solubilization, the fungus was inoculated on Petri dishes containing solid NBRIP (National Botanical Research Institute’s phosphate growth medium), a medium developed for screening phosphate solubilizing microorganisms (Nautiyal, 1999). This medium contains insoluble calcium phosphate (Ca3(PO4)2) as the only source of phosphorus. Five replicate Petri dishes were incubated in the dark at 25°C for 7 days. Positive solubilizing capacity was determined when a visible halo was evident surrounding the fungal colony on agar plates. The pH of the media was adjusted to 7.0 before autoclaving.

Enzymatic activities

Microdochium bolleyi (strain 22-1) was tested for seven hydrolytic capabilities. These were determined on a basal medium (Caldwell et al., 1991) supplemented with the corresponding substrate for each enzyme and agar 1.5%. All tests were performed in triplicate in Petri dishes of 4.5 cm diameter, containing 10 ml of each medium adjusted to pH 6. Plates were incubated for 1-2 weeks at 25°C. Inoculated basal media plates without test substrate and non-inoculated reaction plates were run as controls. Polysaccharide hydrolysis was determined with 1% starch (SIGMA), 1% carboxymethylcellulose (CMC-SIGMA), 1% xylan (SIGMA), 1% apple pectin and 2.4% chitin (SIGMA) as the sole carbon source. The chitin solution was prepared following the protocols described by Hankin & Anagnostakis (1975).

Iodine solution 1% was added to the medium to reveal enzymatic activity for starch, and congo red for CMC and xylan (Teather & Wood, 1982). The reaction was considered positive when a translucent halo formed. Chitin utilization was detected when clear zones were observed around colonies in the opaque agar. Hydrolysis of fatty acid esters was determined by the formation of an opaque halo of calcium palmitate crystals in the basal medium supplemented with 1% Tween 40 (Caldwell et al., 1991). Finally, protein hydrolysis was determined by the formation of a clear halo in basal medium with gelatin 3% as the sole nitrogen source (Gerhardt, 1981).

Metabolic profle analysis

Substrate utilization by M. bolleyi was tested using pre-made FF microplates (Biolog® catalog #1006) containing 95 different carbon and nitrogen sources. For inoculum preparation, M. bolleyi was cultivated in MEA at 25°C. After 1 week the conidia were collected and suspended in a tube containing 5 ml of sterile FF-IF broth (0.25% Phytagel® and 0.03% Tween 40 in DI water). Fungal growth was estimated by measuring turbidity at 750 nm and 490 nm and using the formula: Mycelial Growth = [(abs x 490 nm- abs A1 490 nm) - (abs x 750 nm- abs A1 750 nm)] * 1000. Measurements were made at day 7. Results were graphically presented through a histogram.

Resynthesis and colonization

The resynthesis assay was performed with the identifed strain of M. bolleyi (strain 22-1). Five pots of 200ml were flled with an autoclaved mixture of soil: vermiculite: perlite in 1: 1: 1 (v/v/v) proportion. Hypochlorite (5%) surface-sterilized wheat seeds were sown in the substrate and inoculated with three 1x1-cm plugs with fungal mycelium, taken from the hyphal edge of a 10-day-old fungal colony culturing on MEA. In order to visualize the fungal root colonization, the plants were harvested after 30 days and the roots were processed following the technique developed by Phillips & Hayman (1970) and observed with a light microscope (LM) at 40x magnification.

Ultra-structural studies

To complement the interaction studies, colonized roots were studied with a transmission electron microscope (TEM). Wheat seedlings cultivated on Murashige-Skoog (MS-Sigma) medium (pH 5.8 at 25°C) were inoculated with M. bolleyi. Thirty days later, segments of roots were cut and washed in phosphate buffer, then fxed in 0.25% glutaraldehyde and 4% paraformaldehyde in phosphate buffer 0.1 M (pH 7.4) for 18h at 4°C, rinsed in phosphate buffer under light vacuum and post-fxed in buffered 2% osmium tetroxide for 2h at 4°C, dehydrated in an ethanol-acetone series and included in Durcupan epoxy resin. Thin sections (0.5 µm) were cut with a manual ultramicrotome (Sorvall MT1); these sections were stained with toluidine blue (0.05% [w/v] in benzoate buffer, pH 4.4, for 45 s) and observed with a Zeiss EM 109T transmission electron microscope (service of the Faculty of Medicine (UBA).

results

Identity

The morphology of strain 22-1 agrees well with the description of M. bolleyi (phylum Ascomycota, Pezizomycotina, Sordariomycetes, Xylariomycetidae, Xylariales, Microdochiaceae) provided by Hernández-Restrepo et al. (2016). In pure culture, young colonies were smooth and white-pink in color (Fig. 1A) but later, a melanin-pigmented zone gradually expanded outwards from the colony center. This pigmentation was correlated with the formation of chlamydospores that covered the entire Petri dish after 2-3 weeks. M. bolleyi (strain 22-1) produced crescent-shaped conidia arising from denticulate loci on hyaline septate hyphae (Fig. 1B). The globose or sub-globose conidiogenous cells described by de Hoog & Hermanides-Nijhof (1977) were occasionally observed. In the phylogenetic analysis, strain 22-1 was grouped with members of M. bolleyi (100% of bootstrap support) (Fig. 2). The ITS sequence was submitted to NCBI GenBank with the accession number KF600798.

Fungal physiological characterization

Indole production was evident due to the turning of the Salkowski's chromogenic reagent from in the BIOLOG® microplates, the histogram displayed fve mycelial growth ranges, from R1 (less used substrates) to R5 (more used substrates). According to the histogram, only a few substrates (9, R5) were well used by strain 22-1 (Fig. 3). The most used ones were Adenosine-5-Monophosphate, Putrescine, Glucose-1-Phosphate, 2-Aminoethanol, Glycyl-L-Glutamic Acid, D-Saccharic Acid, L-Serine, L-Ornithine, Adenosine; and the least used ones were B-Cyclodextrin, D-Melibiose, a-D-Lactose, Maltitol, i-Erythriol, m-Inositol, Lactulose, a-Cyclodextrin and Glucoronamide (see attached information for further data). (Table).

 


Fig.1. Microdochium bolleyi. A: Colony general appearance of strain 22-1 grown on MEA at 25ºC for 7 days. B: Hyaline unicellular conidia on cylindrical conidiogenous cells. The scale bar represents 10µm.

 


Fig. 2. Strict consensus of the most parsimonious trees resulting from the ITS data matrix analysis. Numbers above branches refer to bootstrap values.

 

Resynthesis and ultra-structural studies

In LM observations, the isolated strain 22-1 showed typical structures of DSE fungi, colonizing the intercellular spaces of wheat root cortical tissue with hyaline hyphae that did not stain with trypan blue. Melanized and compacted chlamydospores formed inter- and intracellularly in cortical cells and also were frequently found inside the root hairs, occupying the entire space thereof (Fig. 4A-B), but no colonization of the vascular cylinder

or signs of tissue necrosis or disorganization were observed. TEM observations revealed that the hyphal penetration occurred directly at the junction of the epidermal cells, without formation of appressoria. Abundant hyphae in epidermal cells and intercellular space were not associated with the development of papillae or degradation of the plant cell wall (Fig. 4C-D); electronically dense material was observed around these intercellular hyphae.

discussion

In Argentina, DSE of crops are not well-known yet, their identities are poorly understood, and even less their interaction with cereal species (Rothen et al., 2017). In this work, a strain of a DSE fungus, morphologically identified as M. bolleyi, was isolated from wheat roots in agricultural soils in the province of Buenos Aires. Through a phylogenetic analysis, we unequivocally confrmed its identification. Microdochium bolleyi is frequently isolated from cereals and grasses in diverse environments (Mandyam et al., 2010; Wirsel et al., 2001). Although some studies have shown that this fungus produces weak pathogenic effects (Waller, 1979; Kirk & Deacon, 1987; Hong et al., 2008), it is not considered a pathogenic species and it is even able to control take-all diseases (Fox-Roberts & Deacon, 1988), behaving like a commensalist or as a fungus promoting plant growth (Dawson & Bateman, 2001; Liljeroth & Bryngelsson, 2002; Mandyam et al., 2010; Zhang et al., 2008).


Fig. 3. Histogram representing the absolute frequencies (AF) of substrates used by M. bolleyi for fve mycelial growth ranges (R1 to R5). The valúes of mycelial growth were calculated using the following formula: Mycelial Growth = [(abs x 490 nm- abs A1 490 nm) - (abs x 750 nm- abs A1 750 nm)] * 1000.

 


Fig. 4. Microdochium bolleyi colonization of wheat roots observed by optical microscope (A-B) and TEM (C-D). A: Chlamydospore inside a cortical root cell, scale bar 10µm. B: Chlamydospore inside a root hair, scale bar 10µm. C-D: Hyphae colonizing the intercellular space in the cortical tissue (*) with surrounding electrically dense material, scale bars 1 µm.

Physiological characterization

The isolated strain of M. bolleyi did not solubilize P when calcium phosphate was used as an insoluble

phosphorus source. The capacity to solubilize P, though, has been shown in others DSE species, using solid and liquid media with three phosphorus sources: calcium, aluminum and iron phosphate (Priyadharsini & Muthukumar, 2017; Spagnoletti et al., 2017). For instance, Aspergillus ustus, considered as a DSE fungi (Barrow & Osuna, 2002), and Curvularia geniculata (Priyadharsini & Muthukumar, 2017) promote plant growth due to their ability to solubilize P from unavailable phosphate. Further research is needed on this aspect of strain 22-1, using more sensitive methods and different sources of phosphorus.

When considering the enzymatic activities, Cadwell et al. (2000) and Mandyam & Jumpponen (2005) observed a large battery of hydrolytic enzymes in some strains of DSE fungi. In particular, for other Microdochium species, Mandyam et al. (2010) detected the presence of amylase, cellulase, tyrosinase and gelatinase. The strain of M. bolleyi studied here showed positive results just for the amylase test and for a few of the carbon and nitrogen sources evaluated. Some authors have suggested that the capability of DSEs to allow hosts roots to access mineralized nutrients is related to the saprophytic ability (Upson et al., 2010; Usuki & Narisawa, 2007). The few enzymatic abilities and the low proportion of organic compounds used would show a certain limitation by this fungus to mineralize organic sources.

The strain studied produced indole in liquid medium, as has been described for other DSE fungi by Lahlali et al. (2014) and Berthelot et al. (2016), among others. Some authors suggest that the production of microbial IAA would be of great importance for the establishment of symbiosis (Hilbert et al., 2012; Tranvan et al., 2000), while others propose that microbial IAA may induce the growth of plants and could confer improved stress tolerance (Kazan & Manners, 2009; Shi & Chan, 2014). Further tests are required, under different nutritional and stress conditions, to determine how the production of IAA by M. bolleyi may affect its interaction with the host. Interaction with wheat

By means of the re-synthesis, we confrmed that the strain studied produced typical DSE structures (Sieber & Grünig 2013). Unlike many of these endophytes, its hyphae showed little or no melanization at all, with melanin being only found in chlamydospores. This characteristic of the intraradical mycelium has been observed in one strain of Microdochium sp. (Kageyama et al., 2008), Phialophora graminícola (Newsham, 1999), Phialocephala fortinii, and some other DSE as well. Barrow & Aaltonen (2001) consider that the poor melanization of the hyphae in DSE fungi can be attributed to a more active metabolic state in relation to the possible exchange of nutrients with the host, since they observed that melanized hyphae were more extensive in roots of dormant or inactive Atriplex canescens plants, while hyaline hyphae were most abundant in roots of physiologically active plants. Murray &

Gadd (1981) examined further the morphology of an isolate of M. bolleyi and observed the same pattern of colonization in barley, where the roots remained healthy despite being highly colonized. These authors hypothesize that the asymptomatic colonization is due to the prevalence of chlamydospores chains, which are structures of resistance with low metabolic activity. Moreover, intercellular hyphae colonized mainly the cortical parenchyma and root hairs without reaching the vascular cylinder, an aspect that has often been related to non-phytopathogenic fungi (Mandyam & Jumpponen, 2005; Peterson et al., 2008). The pattern of colonization and the lack of typical defense response of host tissues (Morita et al., 2003; Shimizu et al., 2005) together with the absence of any visible disease symptoms make strain 22-1 a "true endophyte" (Mostert et al., 2000).

There are very few studies that analyze the DSE plant-fungus interaction at the ultrastructural level (Peterson et al., 2008, Tsuneda et al., 2009). Here, no defense responses were found when the interaction between T. aestivum and M. bolleyi was studied with TEM, only an electronically dense material was observed around the intercellular hyphae. This is similar to fbrillar material observed surrounding the hyphae in Chinese cabbage in the presence of Heteroconium chaetospira (Yonezawa et al., 2004) and in Asparagus officinalis inoculated with P. fortinii (Yu et al., 2001). Although in the current study the fbers were not observed, it could be due to an inefficient fxation of the material.

conclusion

In Argentina, few studies have focused on the relationships between plants and DSE. In this work, a strain of M. bolleyi isolated from wheat roots was characterized. This fungus produced IAA, it did not grow in most of the sources of P and N, and only amylase activity was detected, suggesting it has low capacity to mineralize organic sources. When M. bolleyi colonized the roots, it formed typical DSE fungal structures and behaved like a "true endophyte" under the conditions evaluated. Further studies are necessary to broaden the knowledge of its role in association with wheat.

 

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al. - Microdochium bolleyi as a DSE fungus

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Recibido el 3 de noviembre de 2017, aceptado el 21 de junio de 2018. Editor: Leopoldo Iannone.



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