Mycelium isolation

Vegetable tissue samples were collected from rubber tree stumps grafted with the IAN-873 clone with rot symptoms in a nursery in Martínez de la Torre (20°05'57.88" N, 97°04'52.3" W), Veracruz, Mexico . The samples were placed on brown paper and inside sealed plastic bags for analysis in the Phytobacteriology Laboratory of the Colegio de Postgraduados, Montecillo Campus.

The mycelium isolation was carried out from fungal structures associated with the tissue with the symptoms of rot disease. From each sample, pieces of the sporome aroind 0.5 cm² were cut and disinfested with sodium hypochlorite (NaOH) at 1.5 % for 1 min, followed by three washes with sterile distilled water. The disinfected pieces were seeded in Malt Agar Extract (MEA) culture medium and incubated at 28 ± 1 °C for 72 h. Mycelium was obtained in axenic culture and preserved in slants in MEA culture medium and sterile mineral oil for subsequent studies.

Morphological description

Partially developed basidiomas were collected from stumps of rubber trees with rot symptoms for microscopic measurement of fungal structures, as well as those grown in the MEA medium; the identification was based on the taxonomic keys elaborated by ^{Olivo and Herrera (1994)} for Schizophyllum species.

Genetic characterization

For the genetic characterization, a colony of mycelium from an axenic culture of eight days of growth in MEA medium of the fungus isolated and identified in this study with the code MZVMT_01 was used. DNA extraction was carried out by the modified AP method (^{Sambrook and Russell, 2001}). The ITS region was amplified with the universal primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3 ') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (^{White et al., 1990}), which are located in the conserved regions of the 18S genes and 28S, respectively. The PCR reactions were carried out in a volume of 25 µL, with 2 µL of DNA at 50 ng µL^-1 and 1 µL of each of the primers at a concentration of 10 µM; PCR buffer 0.5x final concentration; MgCl₂ at 1.25 mm; dNTP's at 0.2 mm and 0.1 U µL^-1 of Taq DNA Polymerase (Promega). The amplification was carried out in a T100 thermal cycler (Biorad) under the following conditions: initial denaturation temperature at 94 °C for 3 minutes, 35 cycles of 94 °C for 30 seconds, alignment of 58 °C for 30 seconds and an extension of 72 °C for 1 min; final extension at 72 ºC for 7 minutes and conservation at 4 °C. The amplified fragments were visualized by 1 % agarose gel electrophoresis, with 0.4 µL of ethidium bromide for 50 minutes at 90 V. Gels were visualized in an Infinitity-ST5 Vilber Lourmat photodocumenter.

The PCR product was purified with the WIZARD^® SV Gel kit and the PCR Clean-Up System, according to the manufacturer's specifications (^{Promega Corporation, 1999).} The purified PCR products were sequenced at Macrogen Korea in Seoul, Republic of Korea.

The DNA sequences were edited and assembled to create the consensus sequence of the amplified regions with the BioEdit Sequence Alignment Editor version 7.0.5.3 program. The assemblies were used to perform a similarity search using BLAST (Basic Local Alignment Search Tool) in the nucleotide database of the National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/Blast). For the phylogenetic analyzes, the consensus sequences were aligned with the clustal method with the CLUSTAL OMEGA 1.2.2 program (^{Sievers and Higgins, 2014}) and the search for the best nucleotide substitution model for each of the species was carried out with the program ModelTest-NG (^{Darriba et al., 2019}). Phylogenetic reconstruction was performed with Bayesian inference using Markov Chains Monte Carlo (MCMC), implemented in the BEAST v1.10.4 program (^{Suchard et al., 2018}) with different generations until the chains stabilized. For the best tree annotation, 25 % of the trees produced were discarded and the subsequnt probability was determined with the remaining trees.

Morphological description

The sporomas observed in the stumps of H. brasiliensis with rot were grouped, small in size, between 0.5-2 cm wide, without stipe with an irregular shell shape, with white to grayish mycelium. The sporoma was gymnocarpal with the sporiferous part limited only to the lower part of the lid and the hymenophore limited to the lower part. The culture of the sporomas in the MEA medium developed colonies of white to cream color, cottony, with branched and irregular growth, of smooth texture and rough surface. Microscopic analysis of the sporomes identified the fungus associated with stump rot of H. brasiliensis as Schizophyllum commune Fries (1815). From the last taxonomic scrutiny, it is classified (^{Kirk, 2020}) as:

Kingdom: Fungi

Phylum: Basidiomycota

Class: Agaricomycetes

Order: Agaricales

Family: Schizophyllaceae

Genus: Schizophyllum

Species: S. commune

Genetic identification

Genetic analysis confirmed the morphological identification of the fungus associated with H. brasiliensis stump rot. The PCR products with the ITS4 and ITS5 rDNA primers amplified an approximate 600 bp fragment of the MZVMT_01 strain morphologically identified as S. commune (Figure 3). ^{Buzina et al. (2001)} obtained amplifications with these same primers in the 660 bp range for S. commune isolates (Figure 3).

M = 1kb molecular marker, lane 1 to 3 samples of Schizophyllum commune; (+) = Positive control; (-) = Negative control (Nuclease-free water).

Figure 3 Amplification of the PCR product of Schizophyllum commune Fr. associated with stump rot of Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. 

The search for similarity analysis by BLAST identified Schizophyllum commune with a maximum identity of 99.47 % with sequences deposited in the NCBI (accession number MN781967 and EU853847).

The phylogenetic tree of Schizophyllum commune was generated from 2 000 000 generations. Phylogenetic reconstruction showed that the MZVMT_01 isolate clustered in the same clade with the S. commune sequence MN781967 (Figure 4), which corresponds to a strain in a study of wood decay fungi in Northwest Arkansas forests (^{Alshammari and Stephenson, 2018}).

Figure 4 Phylogenetic consensus tree based on Bayesian inference illustrating the relationship of Schizophyllum commune Fr. associated with Hevea brasiliensis (Willd. Ex A. Juss.) Müll. Arg. stump rot in Mexico. 

S. commune is a cosmopolitan fungus with wide distribution on all continents and is associated with the colonization of woody tissue and wood rot. It was previously considered a weak pathogen and was considered more of a saprophytic microorganism related to the decomposition of branches, dead wood and stored wood. However, it is now recognized as an important emerging pathogen in plants (^{Schmidt and Liese, 1980}; ^{Ohm et al., 2010}; ^{Takemoto et al., 2010}) and humans (^{Chowdhary et al., 2013}; ^{Saha et al., 2013}; ^{Michel et al., 2015}).

S. commune is included in the group of pathogens that cause the “white rot” disease, among which ^{Schwarze et al. (2000)} have stated that it harbors the greatest variety of degradation mechanisms; it is considered an omnivorous pathogen that invades living tissue and is highly aggressive among basidiomycetes (^{Takemoto et al., 2010}).

The host range includes at least 150 genera of timber, coniferous, fruit, and ornamental trees; in Asia more than 260 species in 150 plant genera are identified as natural hosts and only in Japan 32 species in 22 woody plant genera (De Jong et al., 2006 cited by ^{Ohm et al., 2010}; ^{Takemoto et al., 2010}). Interaction with other pathogens has been demonstrated in young apple trees (Malus sp.) and propagation material that causes white root rot (^{Havenga et al., 2019}).

Genome sequencing of S. commune revealed unique characteristics and increased numbers of genes among other basidiomycetes that code for the production of enzymes that degrade pectin, cellulose, hemicellulose and a unique mechanism to degrade lignin (^{Ohm et al., 2010}). Other studies determined the high production of xylanase, laccase, cellulase, esterases and peroxidase (^{Schmidt and Liese et al., 1980}; ^{Špániková et al., 2007}; ^{Hirai et al., 2008}). Likewise, among 75 basidiomycetes analyzed, S. commune showed greater pectinolytic activity due to the production of polygalacturonases (^{Xavier et al., 2004}). Therefore, it is postulated that S. commune is capable of degrading practically all the components of the cell wall in woody tissue cells (^{et al., 2010}).

Studies on the colonOhm ization of S. commune showed that it invades the woody tissue through the lumen vessels, tracheids, woody vessels, fibers and xylem where cellulose, hemicellulose or pectin are used as carbon sources for its development and subsequent use of lignin and polysaccharides contained in woody tissue (^{Ohm et al., 2010}; ^{Padhiar and Albert, 2011}; ²⁰¹²).

The worldwide distribution of S. commune indicates that it is one of the fungi that adapts to a wide variety of conditions, since it inhabits both temperate and tropical climates. It is stated that the degree of severity of white rot depends on the host species, environmental conditions and growth rate of the fungus; temperature, high relative humidity, global warming and pH have been associated among the most important environmental conditions that influence the aggressiveness of this pathogen (^{Schmidt and Leise, 1980}; ^{Takemoto et al., 2010}). This adaptation to diverse climate and host conditions suggests that genetic variability may exist; this was demonstrated among populations of S. commune that formed different lineages with a close relationship with the geographic origin in North America and Europe; and a more recently expanding lineage in the Caribbean (^{James et al., 1999}; ²⁰⁰¹). The above could suggest that it is also possible that there are differences in virulence and aggressiveness between populations as a phytopathogen; however, there are still no studies documenting these differences as a plant pathogen.

S. commune was recorded in Mexico, in the humid tropics with annual rainfall levels exceeding 2,500 mm (^{Carreño-Ruíz et al., 2019}). In the state of Tabasco, Ficus benjamina L., which also produces latex, has been recognized in fallen wood in Centro, Huimanguillo, Tacotalpa, Teapa, Tenosique and Macuspana municipalities (^{Olivo and Herrera, 1994}; ^{Carreño-Ruíz et al., 2019}). These climatic conditions related to the presence of S. commune exist in Mexico in several rubber-producing localities in the states of Chiapas, Oaxaca, Tabasco, Veracruz and Puebla, with hot humid climates with abundant rains in summer or all year round and ranges of annual precipitation between 1 900 and 4 500 mm per year (^{Inegi, 2009}).

The dissemination of S. commune occurs mainly due to the abundant dispersal of basidiospores by air, which colonize the woody tissue, mainly in young trees; injuries to tissue due to cold, frostbite and sunburn are the main routes of entry and infection (^{Takemoto et al., 2010}). Likewise, pruning, improper pruning, poor nursery management and fertilization practices promote a high incidence of S. commune (^{Snieðkienë and Juronis, 2001}); the wide dispersal ability and prevalence of S. commune up to four years have been documented by ^{Badalyan et al. (2002)}.

In Hevea brasiliensis, the basidiomycete Rigidoporus microporus (Sw.) Overeem (1924) has been identified as a causal agent of “white rot” in roots (^{Oghenekaro, 2016}), but not S. commune. In the interaction of Hevea brasiliensis with S. commune, the little information available is limited to the study of the association of this basidiomycete as part of the saprophytic community in the decomposition of fallen branches and stored wood (^{Hong, 1982}; ^{Seephueak et al., 2011a}; ^2011b), but not as a parasite and pathogen in plants. The high genetic uniformity in the rubber crop, the nursery management and the climatic conditions could be important factors in the establishment, dispersal and aggressiveness of S. commune in Hevea brasiliensis in Mexico.