rmfi Revista mexicana de fitopatología Rev. mex. fitopatol 0185-3309 2007-8080 Sociedad Mexicana de Fitopatología A.C. 10.18781/R.MEX.FIT.2408-1 Scientific Article Characterization of Sclerotium rolfsii and species of Trichoderma in commercial crops of potato in Sonora and Sinaloa, Mexico Irazoqui-Acosta María Belén 1 Felix-Gastelum Rubén 1 * Leyva-Madrigal Karla Yeriana 1 Longoria-Espinoza Rosa Maria 1 Herrera-Rodríguez Gabriel 2 Armenta-López Sara Elodia 2 Universidad Autónoma de Occidente, Unidad Los Mochis, Departamento de Ciencias Naturales y Exactas, Boulevard Macario Gaxiola y Carretera Internacional, S/N, Los Mochis, Sinaloa, México. CP. 81223. Universidad Autónoma de Occidente Universidad Autónoma de Occidente Los Mochis Sinaloa 81223 Mexico Junta Local de Sanidad Vegetal del Valle del Fuerte. Lázaro Cárdenas, 315 Pte. Col. Centro, Los Mochis Sinaloa, CP. 81200. México. Junta Local de Sanidad Vegetal del Valle del Fuerte Junta Local de Sanidad Vegetal del Valle del Fuerte Los Mochis Sinaloa 81200 Mexico *Autor de Correspondencia: Rubén Félix-Gastelum ruben.felix@uadeo.mx 30 09 2025 Sep-Dec 2025 43 3 rmexfit24081 09 08 2024 22 08 2025 This is an open-access article distributed under the terms of the Creative Commons Attribution License ABSTRACT

Background/Objective

. Sclerotium rolfsii causes losses in potato production up to 20% in the states of Sonora and Sinaloa. No studies exist supported by the scientific method on the identification of the fungus in the crop, neither of the species of Trichoderma present in the soil with potential to control the disease under field conditions. The objectives of the present study were to: a) obtain isolates of S. rolfsii from soil subject to the potato crop in the states of Sonora and Sinaloa, and determine the population density of sclerotia of the fungus; isolate and quantify the population density of Trichoderma spp., in the same soil to use them in subsequent studies for controlling the disease and determine the pathogenicity of S. rolfsii in potato tubers under laboratory conditions.

 Materials and Methods.

The isolates were collected from 239 commercial potato fields in the states of Sonora and Sinaloa. The population density was determined by counting the sclerotia of S. rolfsii and the colony forming units (CFU g-1) of Trichoderma in soil. The identification of the fungi was performed by morphological studies and molecular techniques including the internal transcribed spacer (ITS) for S. rolfsii and the subunit of RNA polymerase II (RPB2) for Trichoderma spp. The pathogenicity tests of the isolates of S. rolfsii were determined in potato tubers var. Fianna in the laboratory.

 Results

. Twenty isolates of S. rolfsii were identified whose population density varied from 2 to 24 sclerotia kg-1 of soil. On the other hand, 26 isolates of Trichoderma spp. were identified; 16 of them corresponded to T. asperellum, five

to T. asperelloides, four to T. afroharzianum and one to T. azevedoi with a population density from 2 to 8 CFU g-1 of soil. The isolates of S. rolfsii resulted pathogenic to potato tubers var. Fianna, with different levels of aggressiveness.

 Conclusion.

The results obtained in the present study indicate the presence of S. rolfsii in 8.4% of the 239 potato growing fields included in the study sampled in the growing cycles 2019-2022 con population densities from 2 to 24 sclerotia g-1 of soil. The pathogen confirmation by conventional and molecular techniques, as well as its capability to induce soft rot of potato tubers var. Fianna, provides evidence of its potential impact on potato production. Variable aggressiveness was detected among isolates, highlighting the Scr4 isolate as the most aggressive with penetration up to 16.9 mm and soft rotting of 10.5%. In parallel, 26 isolates of Trichoderma spp. were identified in 10.9% of the fields, with population densities from 2 to 8 CFU g-1 of soil. These findings highlight the importance of incorporating sustainable strategies for disease management, considering both the variability of the pathogen and the possible application of native antagonists; also, it is stressed the necessity to investigate the population dynamics of the pathogen and agricultural practices to minimize its survival.

 Introduction

The potato (Solanum tuberosum) is considered one of the most important crops, due to its worldwide production, after wheat (Triticum spp.) and rice (Oryza sativa) (FAO, 2024). In Mexico, potato production is estimated in 1,986,198 t (SIAP, 2023), with the states of Sonora and Sinaloa being the main producers, and in which the Fianna, Orquesta, Atlantic, FL-1867, FL-2027, FL-2395 and FL-2312 varieties stand out, accounting for 52.4% of the country's production (SIAP, 2023). Yield is determined by diverse factors in crop management, the most outstanding of which include diseases of biotic origin, which affect plant development and the quality of the tubers (Herrera and Scott, 1993). The fungi, bacteria, nematodes and viruses that affect the tuber produce foliar spots, galls, mosaics, rots and others (Fiers et al., 2012).

Soft rot is a disease caused by the fungus Sclerotium rolfsii. The symptoms consist of maroon lesions in the stem and tubers, and in advanced stages of the disease, soft rot is found, with slightly sunken lesions. Signs are characterized by the development of white mycelia and the presence of round maroon sclerotia, between 2 and 4 mm in diameter on the infected tissue (Garibaldi et al., 2007; Roca et al., 2016). Losses caused by the disease have reached up to 60% in other parts of the world (Haque and Khan, 1977). In Sinaloa, damages have been estimated in 20% in commercial potato plantations.

S. rolfsii is a necrotrophic fungus that lives in the soil, with a high capacity of saprophytic growth. It is considered a pathogen of high economic importance in agriculture, due to its wide range of hosts, wide geographic distribution and ability for survival due to its formation of sclerotia and mycelia, allowing it to survive in dead organic matter or alternative hosts (Aycock, 1966; Punja, 1985).

In recent years, in the states of Sinaloa and Sonora, symptoms of soft rot have been found in potato crops. It is worth mentioning that studies have been carried out for the control of this disease, in which the adequate doses of fungicide have been determined for its control (Browne et al., 2002). However, the high cost, environmental regulations and the toxicity of the fumigants limit the use of this type of molecules in the management of this disease (García-González et al., 2022). The application of Trichoderma species as biocontrol agents has also been used, and they have proven to be effective (Guzmán et al., 2014; Zúñiga and Ceja, 2017). Martínez-Martínez et al. (2020) carried out a study in Mexico, in which different strains of different species of Trichoderma were used against the mycelial growth of S. rolfsii, resulting in a viable alternative, both in vitro and in planta for the control of this phytopathogen in chickpea. Nevertheless, no research has been carried out in Mexico on this disease on potato crops.

Due to this, the aims of this study were: a) to obtain S. rolfsii isolates from soils under potato cultivation in the states of Sonora and Sinaloa, and to determine the population density of the fungal sclerotia; b) to isolate, quantify the population density and morphologically and molecularly identify Trichoderma spp. isolates from the same soils in order to use them in subsequent studies for disease control; and d) to determine the pathogenicity of S. rolfsii in potato tubers under laboratory conditions.

 Materials and Methods

Gathering samples. During the development of the study, soil samples were obtained from 239 plots in which potato is produced in Sonora and Sinaloa, in the 2019-2020, 2020-2021 and 2021-2022 autumn-winter growing seasons. Ten-hectare plots were selected from which 500 g subsamples were collected using the method known as “five-point sampling”. The samples were homogenized, taking 1 kg as a final sample.

Population density, isolation and morphological characterization of S. rolfsii isolates. In order to determine the S. rolfsii populations, sclerotia were extracted from the soil samples using the Fenwick can technique (Van-Bezooijen, 2006). A soil sample weighing 500 g was placed in a number 20 (850 µm) sieve, on the can. In the water output, a number 60 sieve was placed (250 µm). Subsequently, the retained particles were dried in the shade over absorbent paper for 24 h. The particles were observed under a stereoscopic microscope (Carl Zeiss®; SteREO Discovery.V20, Germany); the sclerotia obtained in the sample were then collected and quantified. Next, they were disinfested in a 1% sodium hypochlorite solution for one minute and planted in a Potato Dextrose Agar (PDA) culture medium (BD Bioxon®) supplemented with chloramphenicol (15 μg mL-1) (Armenta-López et al., 2021). The dishes were incubated at 27 °C for 48 h (Yamato Scientific Co., LTD®; Economy Incubator IN804, Tokyo, Japan). From the growth point of the colony, a disk with 5 mm in diameter was taken and transferred into Petri dishes containing PDA and then incubated at 27 °C.

For the morphological identification of Sclerotium spp., a PDA disk, 5 mm in diameter and with active growth from each isolate, was placed on the edge of three 90 mm Petri dishes with the same medium. The dishes were incubated at 27 °C until the fungus filled the dish. To determine the mycelial growth rate, the following formula was used: TC = (Final growth - Initial growth) / time of incubation (Guigón-López et al., 2010). The colonial morphology was described: color of colony in the dish (front and back); mycelium shape and type. The characteristics of the hyphae and the formation of the fibulae and the color of the sclerotia were also recorded (Paparu et al., 2020). Additionally, the average number of sclerotia produced was evaluated and the size of 30 of them was determined per Petri dish in each one of the isolates (Prasad et al., 2012).

Population density, isolation and morphological characterization of Trichoderma spp. isolates. In order to obtain Trichoderma spp. isolates, 0.5 g of soil from each sample were weighed (Ohaus Corporation®; YS600, China), sprinkled over the surface of a Petri dish with PDA and incubated for 10 days at 27 °C. Once the colonies with a typical morphology for the genus developed (Gary and Prakash, 2015), they were quantified in each Petri dish, transferred into dishes with the same medium and incubated at 27 °C. The Trichoderma colonies were purified using the monosporic culture technique by Estrada et al. (1997). From every Trichoderma isolate, two 5-mm mycelium disks were transferred to PDA, Spezieller Nährstoffarmer Agar (SNA) (modified by Nirenberg, 1976) and Corn Meal Agar (CMA) (BD BBL®), on the edge of Petri dishes, 90 mm in diameter (three replications for every medium). The dishes were incubated for seven days in a bioclimatic chamber (Thermo Scientific®; Precision 3759, USA), using cold white fluorescent light at 25 °C (Pacheco et al., 2016). Mycelial growth was measured every 24 h until it covered the Petri plates with PDA and SNA. Measurements were taken from the edge of the inoculum disk to the most distant point of the colony. The previously described formula was used to determine the growth rate (Guigón-López et al., 2010). In the PDA and SNA media, the color and edge of the colony were evaluated, whereas in CMA, pustule size, type of development, shape of the conidiophores, the position of the chlamydospore, and shape and size of the phialides and conidia were determined. The structures were mounted on slides using the adhesive tape and glycerin technique (Gary and Prakash, 2015) and observed under a compound microscope with a 100x lens (Carl Zeiss®; Axio Imager M2, Germany). The shape and size (μm) of 30 phialides and 30 conidia were recorded for each isolate.

Molecular identification of S. rolfsii and Trichoderma spp. Three disks of each isolate were placed in centrifuge tubes containing 25 mL of nutrient broth. The cultures were incubated at 27 °C and constantly shaken for five days at 150 rpm (Labnet International, Inc.®; Labnet Incubator Shaker 311DS, USA). The mycelium was placed in 2 mL Eppendorf tubes. The genomic DNA was obtained with the CTAB method at 2% (Sanger et al., 1977) and finally adjusted to 50 ng μL-1. The presence and integrity of the genomic DNA was verified by electrophoresis (Thermo Scientific®; Electrophoresis Power Supply EC1000XL, China) in 1% agarose gel 1%. The bands were observed using a photodocumentation system (BIORAD®; Universal Hood ll, USA). The DNA concentration and quality was determined in a NanoDrop (Thermo Scientific®; NanoDrop One ND-ONE, USA).

The molecular identification of the Sclerotium isolates was carried out with the Polymerase Chain Reaction (PCR) to amplify a fragment of 670 ± pb of the Internal Transcribed Spacer (ITS) with oligonucleotides ITS1 (5´ TCC GTA GGT GAA CCT TGC GG 3´) and ITS4 (5´ TCC TCC GCT TAT TGA TAT GC 3´) (White et al., 1990).

Oligonucleotides fRPB2-5F (5’ GAT CGA TCA CGA TGA TCA TCT TTC GG 3’) and fRPB2-7cR (5’ CCC ATA GGC TTG TCT TAG CCC AT 3’) (Liu et al., 1999) were used

to amplify a 1100 ± pb fragment of the subunit of polymerase II RNA (RPB2) from Trichoderma (Chaverril et al., 2003). The reaction mixture for each sample was 25 µL, which contained 1X buffer, 0.2 µg µL-1 BSA, 0.2 mM dNTP´s, 1.5 mM MgCl2, 0.5 µM of each oligonucleotide, 0.1 U µL-1 of Taq polymerase (Promega®) and 50 ng of ADN. The final volume was adjusted with ultrapure water. The conditions of amplification were as follows: initial 94 °C denaturation for 5 min, 35 denaturation cycles at 94 °C for 30 seg, alignment (57 °C for ITS and 54 °C for RPB2) for 40 sec and a 72 °C extension for 45 seg, followed by a final extension at 72 °C for 5 min in a thermocycler (BIORAD®; C1000 Thermal Cycler CFX96, Singapore). A total of 5 µL of reaction product and 1X loading buffer (Promega®) were used to perform electrophoresis in 1.5% agarose gel. The amplified fragments were observed using the photodocumentation system and sent for sequencing to Macrogen® Seoul, Korea.

The sequences of the ITS region for S. rolfsii and the RPB2 gene for Trichoderma were edited in the BioEdit program, version 7.2.5 (Hall, 1999) and compared in the GenBank data base, using the BLASTn algorithm. The sequences obtained were aligned with reference sequences, belonging to types strains of different species within the Trichoderma complexes (T. harzianum and T. viride), as well as type strains belonging to Sclerotium rolfsii, using the MUSCLE software (Edgar, 2004) implemented in MEGA X, version

10.2.4 (Kumar et al., 2018). The Ceratobasidium cornigerum (AJ302006) and Protocrea pallida (CBS 121552) sequences were used as an external group in the phylogenetic analysis of Sclerotium and Trichoderma, respectively. The alignment underwent a model- fit test for nucleotide substitution and partitioning scheme in PartitionFinder v1.1.1 (Lanfear et al., 2012), following the greedy algorithm and the Akaike Information Criterion (AIC). Phylogenetic inference for Trichoderma was carried out by Maximum Likelihood (ML) in RAxML v7.2.8 (Stamatakis, 2006) and Bayesian Inference (BI) in MrBayes 3.2.7 (Ronquist et al., 2012), whereas inference for Sclerotium was performed only by ML. In both analyses, the General Time Reversible model with gamma distribution and invariant sites (GTRGAMMAI) and the partitioning scheme defined by PartitionFinder were used. The ML analysis used 1,000 bootstrap replicates, and BI used 2 simultaneous runs of 1 million generations with six Markov and Monte Carlo chains, sampling every 1,000 trees and discarding 20% of trees as burn-in. Run stability and convergence were evaluated in Tracer ver. 1.7.2 (Rambaut et al., 2018). Phylograms were edited in FigTree v1.4.0 (Rambaut, 2014).

Pathogenicity of Sclerotium rolfsii in potato tubers. The pathogenicity of 20 S. rolfsii isolates (Scr2, Scr3, Scr4, Scr5, Scr6, Scr7, Scr8, Scr9, Scr10, Scr11, Scr12, Scr13, Scr14, Scr17, Scr47, Scr48, Scr49, Scr50, Scr51 and Scr54), obtained from soils that had undergone potato plantation in Sonora and Sinaloa was evaluated. Potato tubers var. Fianna were superficially washed and disinfected with 70% alcohol. Five healthy tubers were used for each treatment and placed in moist chambers to achieve high relative humidity. A superficial lesion was created in the tuber with a hole puncher, 5 mm in diameter, and on the lesion, a PDA disk, 5 mm in diameter, was placed with active fungal growth. As a control, tubers were inoculated with PDA disks without the fungus. The inoculated tubers and controls were placed in a bioclimatic chamber at 27 °C (Daami-Remadi et al., 2012).

The data for temperature and moisture inside the moist chamber were recorded with a MadgeTech®; RFRHTemp2000A, Wireless Temperature / Humidity Data Logger, USA once per hour during the experiment.

The pathogenicity and virulence of the isolates in the tubers was determined eight days after the inoculation with the pathogen; for this, the tubers were cut transversally in the center of the lesion and the maximum width (W) and depth (PR) of the lesion were measured to determine the penetration of the pathogen (P), which was calculated using the formula by Lapwood et al. (1984): P (mm) = (A / 2 + (PR - 6)) / 2. The percentage of soft rot of the tuber tissues was also estimated, using the methodology by Bourne et al. (1981) and Hildenbrand and Ninnemann (1994), which consists of weighing each tuber (PT), then extracting the damaged (rotten) tissue and weighing again (PS). To determine the percentage of tissue with rotting, the following formula was used: (%) = (PT - PS / PT) ×

100. Subsequently, the pathogen was isolated from the tissue that displayed characteristic symptoms of the disease, and it was morphologically identified to fulfill Koch's postulates. The experiment was run twice.

Statistical data analysis. The normality of the data on the penetration and percentage of soft rot caused by the pathogen was verified using the Kolmogorov-Smirnov test with the SAS 9.0 statistical package. The analyses showed that the data distribution was not normal, therefore the data was subject to a non-parametric Kruskal-Wallis analysis with a value of (P>0.05).

 Results and Discussion

Population density, isolation and morphological characterization of S. rolfsii isolates. Twenty S. rolfsii isolates were obtained from the same number of fields, during the time of sampling of the 239 plots from Sonora and Sinaloa, which accounted for 8.4% of the plots in which the fungus was found; the population density varied between 2 and 24 sclerotia per kg of soil (Table 1). The isolates presented whitish mycelia with a fibrous and cottonlike appearance, and fan-shaped (Figure 1A). Hialin hyphae, septated, with fíbulae (Figure 1B). Shades ranging from whitish to yellowish were reported on the reverse side of the colonies, similar to what was reported by Paparu et al. (2020). According to Mahadevakumar et al. (2018) S. rolfsii presents different morphological characteristics, in both its colony and in the sclerotia, influenced by the wide range of hosts and weather conditions in which it develops.

The isolates displayed a radial growth of 4-16 mm/day, until they reached a diameter of 90 mm in the Petri dish, between 5 and 8 days of incubation (Prasad et al., 2012). Okereke and Wokocha (2007) reported that the variation in the growth of the isolates is due to differences in the levels of nutrients, ecology or genetic differences.

The sclerotia developed 10 to 20 days after transferring the isolates to the medium in Petri plates, and their diameter varied, between 0.5 and 2 mm. The isolates formed between 18 and 153 sclerotia per Petri dish. The sclerotia displayed a light maroon to dark maroon color, depending on their level of maturation, similar to the morphological structures published by Zhou et al. (2019). Likewise, the size of the sclerotia was similar to those a lo reported by Díaz-Nájera et al. (2018).

Identification, population density and origin of 20 <italic>S. rolfsii</italic> isolates obtained from soils in which potato is grown in Sonora and Sinaloa.
Code Identification Esclerotia/kg of soil Growing season Municipality/State Coordenates GenBank code
Scr2 S. rolfsii 8 2019-2020 El Fuerte, Sinaloa 25.942500 -108.809166 OR514111
Scr3 S. rolfsii 10 2019-2020 Ahome, Sinaloa 25.777500 -109.040277 OR514112
Scr4 S. rolfsii 24 2019-2020 Ahome, Sinaloa 25.701944 -109.043333 OR514113
Scr5 S. rolfsii 6 2019-2020 Ahome, Sinaloa 25.851944 -108.885000 OR514114
Scr6 S. rolfsii 12 2019-2020 Ahome, Sinaloa 25.669722 -109.006111 OR514115
Scr7 S. rolfsii 10 2019-2020 Ahome, Sinaloa 25.769444 -109.045000 OR514116
Scr8 S. rolfsii 20 2019-2020 Ahome, Sinaloa 25.836944 -108.926388 OR514117
Scr9 S. rolfsii 4 2019-2020 Ahome, Sinaloa 25.691944 -109.010277 OR514118
Scr10 S. rolfsii 6 2019-2020 El Fuerte, Sinaloa 25.944917 -108.908333 OR514119
Scr11 S. rolfsii 2 2019-2020 Ahome, Sinaloa 25.743611 -108.998610 OR514120
Scr12 S. rolfsii 4 2019-2020 Ahome, Sinaloa 25.876111 -108.835000 OR514121
Scr13 S. rolfsii 2 2019-2020 Guasave, Sinaloa 25.652500 -108.763888 OR514122
Scr14 S. rolfsii 8 2019-2020 Ahome, Sinaloa 25.789166 -108.895277 OR514123
Scr17 S. rolfsii 18 2019-2020 Ahome, Sinaloa 25.773330 -109.280277 OR514124
Scr47 S. rolfsii 2 2021-2022 Ahome, Sinaloa 25.465355-108.542845 OR514125
Scr48 S. rolfsii 6 2021-2022 Ahome, Sinaloa 25.464949 -108.543231 OR514126
Scr49 S. rolfsii 16 2021-2022 Ahome, Sinaloa 25.465739 -108.541982 OR514127
Scr50 S. rolfsii 10 2021-2022 Ahome, Sinaloa 25.465119 -108.543142 OR514128
Scr51 S. rolfsii 14 2021-2022 Ahome, Sinaloa 25.464987 -108.543004 OR514129
Scr54 S. rolfsii 18 2021-2022 Altar, Sonora 30.945163 -111.830293 OR514130

Regarding the production of sclerotia, the results of this study are consistent with those reported by Ayed et al. (2018); their formation began in the same period recorded by Mahadevakumar et al. (2018). This differs from what was documented by Paparu et al. (2020), who reported the production of sclerotia 28 days after growth on PDA.

Morphological characteristics of Sclerotium rolfsii (Scr17). (<bold>A</bold>) Colony morphology in PDA medium, and sclerotia (red arrow); (<bold>B</bold>) Hyphal hyalin (yellow arrow) and fíbulae (blue arrow) (100x). Scale: (B= 50 µm).

The Maximum Likelihood dendrogram generated with Sclerotium ITS region sequences showed that the 20 isolates obtained from 20/239 potato fields is shown on Figure 2.

Phylogenetic analysis of <italic>Sclerotium rolfsii</italic> from Sonora and Sinaloa. Maximum likelihood tree based on the Internal Transcribed Spacer (ITS) of 20 <italic>S. rolfsii</italic> isolates. The isolates characterized in this study appear in bold letters. The bootstrap values over 50 are shown as percentages. The scale bar indicates the expected number of nucleotide substitutions.

The analysis showed that all 20 isolates: Scr2, Scr3, Scr4, Scr5, Scr6, Scr7, Scr8, Scr9, Scr10, Scr11, Scr12, Scr13, Scr14, Scr17, Scr47, Scr49, Scr49, Scr50, Scr51 and Scr54 belonged to the species Sclerotium rolfsii, as they clustered with the sequences of the type C13 (KY175225), CBS 115.22 (MH854711) and AFTOL-ID (DQ484062) isolates of this

species, with a high bootstrap support (80%) (Figure 2); these results coincide with reports by Mahadevakumar et al. (2018).

Population density and morphological and molecular characterization of Trichoderma species isolates. Twenty-six Trichoderma spp. isolates were obtained from soil from 239 fields from Sonora and Sinaloa, which accounted for 10.9% of the plots with the fungus, with a population density of 2 to 8 Culture-Forming Units per gram of soil (UFC g-1) (Table 2).

Identification ID, population density (CFU) and origin of 26 <italic>Trichoderma</italic> isolates sampled from soil of potato (<italic>S. tuberosum</italic>) commercial fields in Sonora and Sinaloa.
Code Species x CFU g-1 Growing season Municipality/State Coordenates GenBank code
TES19 T. asperelloides 4 2020-2021 Caborca, Sonora 31.041666 -112.331111 OR521159
TES20 T. asperelloides 2 2020-2021 Altar, Sonora 31.062222 -111.834722 OR521160
TAF21 T. afroharzianum 6 2020-2021 Altar, Sonora 30.909444 -111.804444 OR521161
TAM22 T. asperellum 4 2020-2021 Caborca, Sonora 30.541388 -112.273611 OR521162
TES23 T. asperelloides 8 2020-2021 Caborca, Sonora 31.995555- 112.360277 OR521163
TES24 T. asperelloides 6 2020-2021 Caborca, Sonora 31.066660- 112.338333 OR521164
TES26 T. asperelloides 4 2020-2021 Caborca, Sonora 30.660000 -111.270550 OR521165
TAM27 T. asperellum 2 2019-2020 Guasave, Sinaloa 25.722955 -108.740276 OR521166
TAM30 T. asperellum 2 2019-2020 El Fuerte, Sinaloa 25.944916 -108.908333 OR521167
TAM31 T. asperellum 2 2019-2020 Guasave, Sinaloa 25.655277 -108.561944 OR521168
TAF33 T. afroharzianum 4 2020-2021 S. de Leyva, Sin. 25.952500 -108.406388 OR521169
TAM35 T. asperellum 4 2020-2021 Ahome, Sinaloa 25.760550 -109.266388 OR521170
TAM37 T. asperellum 6 2020-2021 Guasave, Sinaloa 25.712500 -108.782222 OR521171
TAF38 T. afroharzianum 8 2020-2021 Guasave, Sinaloa 25.580833 -108.603055 OR521172
TAM57 T. asperellum 8 2021-2022 Ahome, Sinaloa 25.481112 -108.571717 OR521173
TAM59 T. asperellum 6 2021-2022 Guasave, Sinaloa 25.679176 -108.665316 OR521174
TAM64 T. asperellum 4 2021-2022 Ahome, Sinaloa 25.819266 -108.955429 OR521175
TES65 T. asperelloides 4 2021-2022 Ahome, Sinaloa 25.491132 -108.571830 OR521176
TAM67 T. asperellum 2 2021-2022 Ahome, Sinaloa 25.491299 -108.571854 OR521177
TAM68 T. asperellum 2 2021-2022 Ahome, Sinaloa 25.491496 -108.571569 OR521178
TAM69 T. asperellum 2 2021-2022 Ahome, Sinaloa 25.491299 -108.571854 OR521179
TAM70 T. asperellum 4 2021-2022 Ahome, Sinaloa 25.498320 -108.571704 OR521180
TAI73 T. azevedoi 2 2021-2022 Ahome, Sinaloa 25.498320 -108.572148 OR521181
TAM74 T. asperellum 4 2021-2022 Ahome, Sinaloa 25.491445 -108.571659 OR521182
TAF75 T. afroharzianum 4 2021-2022 Ahome, Sinaloa 25.498320 -108.572148 OR521183
TAM76 T. asperellum 6 2020-2021 Caborca, Sonora 31.995555 -112.360277 OR521184

x Species were identified based on morphological and molecular characteristics.

Based on the morphological characteristics, it was determined that the Trichoderma isolates belonged to two species complexes: 21 isolates belonged to the T. viride complex (TES19, TES20, TAM22, TAM23, TES24, TES26, TAM27, TAM30, TAM31, TAM35, TAM37, TAM57, TAM59, TAM64, TES65, TAM67, TAM68, TAM69, TAM70, TAM74

and TAM76) (Table 3; Figure 3) and five, to the T. harzianum complex (TAF21, TAF33, TAF38, TAI73 y TAF75) (Table 3; Figure 4).

The isolates of the T. viride complex displayed a growth rate in PDA of 10-18 mm/day, and in SNA, of 8-14 mm/day (Table 3), similar what Guigón-López et al. (2010) reported. The colony morphology was similar for all isolates in CMA, where the color of the colony varied from light green to dark green as they matured. The edge was wavy and smooth, with a floccose to arachnoid appearance, aqueous white mycelium and hyaline hyphae (Table 3; Figure 3A y B) (Rai et al., 2019). Dark green pustules, 1-2 mm in diameter (Table 3; Figure 3C). Long and branched conidiophores (Table 3; Figure 3D and E). Sub-globose to globose, smooth conidia, 2.5 to 3.8 µm long and 2.0 to 3.0 µm wide (Table 3; Figure 3F). Lageniform-thin phialides with branched ends, 4.2 to 14.3 µm long and 2.6 to 5.0 µm wide (Table 3; Figure 3D y E). No chlamidospores were found (Table 3) (Gary and Prakash, 2015).

On the other hand, the T. harzianum complex isolates displayed a growth rate of 11 to 19 mm/day in PDA and 11 to 17 mm/day- in SNA (Table 3; Figure 4A and B), which coincides with the ranges reported by Gary and Prakash, (2015). In HMA, the pustules were light green in color (Table 3; Figure 4C) (Rai et al., 2019), with sub-globose, smooth conidia, 2.0 to 3.0 µm long and 1.6 to 2.3 µm wide (Table 3; Figure 4H). Ampulliform phialides with a distinctive neck and a narrow tip, 5.8 to 7.2 µm long and 2.5 to 3.0 µm wide (Table 3; Figure 4F and G). Intercalary and terminal chlamydospores were observed (Table 3; Figure 4D and E) (Gary and Prakash, 2015).

Morphological characteristics of isolates of the <italic>Trichoderma viride</italic> and <italic>Trichoderma harzianum</italic> species complexes in three culture media.
Morphological characteristicsZ Culture medium T. viride T. harzianum
Colony Radial mycelial growth (mm/day) PDA 10-18 11-19
SNA 8-14 11-17
PDA
Color SNA Light green to dark green Light green to dark green
CMA
Pustules Size(mm) CMA 1-2 1-2
Mycelium Shape PDA and Floccosase and arachnoid Floccosase and arachnoid
Hyfae color CMA Hyaline Hyaline
Conidium Shape CMA Subglobose, ovoid, Smooth, warty Subglobose, Smooth
Size (length) 2.47-3.78 μm 2.03-2.99 μm
Size (width) 2.02-3.05 μm 1.64-2.30 μm
Conidiophores Shape CMA Long and branched Branched
Chlamidospores Position CMA Absent Terminal and intercalar
Phialides Shape CMA Lageniform and thin Ampuliform
Size (Length) 4.2-14.3 μm 5.8-7.2 μm
Size (width) 2.6-5.0 μm 2.5-3.0 μm

ZThe morphological characteristics were determined in 2- to 3-day old colonies with 12-hour light regimes. In bold the characteristics are more distinctive.

Colony and microscopic morphology of <italic>Trichoderma asperellum</italic> (TES19). (<bold>A</bold>) colony in PDA culture medium. (<bold>B</bold>) colony in SNA culture medium. (<bold>C</bold>) conidial pustules. (<bold>D</bold> and <bold>E</bold>) conidiophores and (<bold>F</bold>) conidia (100x). (<bold>C-F</bold>) in HMA medium. Scale: (C= 2 mm) (D= 10 µm) (E and F= 5 µm). This species belongs to <italic>T. viride</italic> complex.

Colony and microscopic morphology of <italic>Trichoderma afroharzianum</italic> (TAF33). (<bold>A</bold>) colony in PDA culture medium. (<bold>B</bold>) colony in SNA culture medium. (<bold>C</bold>) conidial pustules. (<bold>D and E</bold>) chlamidospores and (<bold>F and G</bold>) conidiophores (<bold>H</bold>) conidia (100x). (C-H) in HMA medium. Scale: (C= 2 mm) (D and H= 5 µm) (E, F and G= 10 µm). This species belongs to <italic>T. harzianum</italic> complex.

The Maximum Likelihood dendrogram of the RPB2 gene for the T. viride complex identified two species among the 16 analyzed isolates. Isolates TAM76, TAM67, TAM30, TAM35, TAM37, TAM57, TAM23, TAM59, TAM27, TAM69, TAM22, TAM31,

TAM70, TAM68, TAM64 and TAM74 were clustered with the sequences from strains GJS 01-15, TR3(T) and GJS 02-63 of Trichoderma asperellum¸ with a bootstrap support of 78% and a 0.99 PP, whereas isolates TES19, TES20, TES26, TES24 and TES65 were clustered with strains GJS 02-63, GJS 04-111(T) and CML 2676 of Trichoderma asperelloides (Bootstrap 98%; PP 1; Figure 5). On the other hand, the phylogram of the T. harzianum complex confirmed that isolates TAF21, TAF38, TAF75 and TAF33 belonged to the species Trichoderma afroharzianum, since they are clustered with the sequences of the strain of this species, CEN1417, CEN1410, GJS 04-186(T) and CBS 466.94, with a high bootstrap support (92%) and a posterior probability (1). On the other hand, isolate TAI73 was identified as Trichoderma azevedoi, since it formed a clade with the strains of this species, CEN1423, CEN1403 y CEN1422(T) (Bootstrap 53% and PP 0.72) (Figure 6). Jaklitsch and Voglmayr (2015) and Athafah et al. (2020) reported three Trichoderma species based on the region RPB2 (T. asperellum, T. asperelloides and T. afroharzianum) obtaining plant materials from forest areas. In turn, Inglis et al. (2020) identified T. azevedoi with the same region, in soil samples gathered from garlic and onion fields in eight different locations in Brazil.

Maximum Likelihood phylogram of the <italic>Trichoderma viride</italic> species complex, based on the RNA polymerase II subunit (RPB2). The 21 <italic>Trichoderma</italic> isolates, obtained from potato plantations in Sonora and Sinaloa characterized in this study, appear in bold and highlighted in color. The bootstrap values >50 and the posterior probability values >0.5 are shown in the nodes. The scale bar indicates the expected number of nucleotide substitutions.

Maximum Likelihood phylogram of the <italic>Trichoderma harzianum</italic> species complex, based on the RNA polymerase II subunit (RPB2). The five <italic>Trichoderma</italic> isolates obtained from potato plantations in Sonora and Sinaloa characterized in this study, appear in bold and highlighted in color. The bootstrap values >50 and the posterior probability values >0.5 are shown in the nodes. The scale bar indicates the expected number of nucleotide substitutions.

Pathogenicity of Sclerotium rolfsii in potato tubers. Eight days after inoculation, all potato tubers of the Fianna variety inoculated with the S. rolfsii isolates displayed symptoms of soft rot (Paparu et al., 2020; Paul et al., 2021) (Table 4) with variations in the levels of virulence between isolates; this piece of information is worth considering when evaluating the genetic

resistance of potato to the disease. Symptoms were similar to the lesions produced by the fungus in the field. The tubers displayed circular and irregular soft rot, with a brown edge (Figure 7), whereas the control tubers remained healthy throughout the experimental period. The penetration of the 20 isolations in the tuber varied from 9.5 to 16.9 mm, with significant differences (P > 0.05). The greatest penetration took place with isolate Scr4 (16.9 mm), whereas isolate Scr9 displayed the lowest penetration (9.5 mm). Isolate Scr7 caused a penetration of 15.2 mm, and displayed no significant differences with isolates Scr17, Scr48 and Scr10, with a penetration of 14.0, 13.7 and 13.6 mm, respectively (Table 4). On the other hand, the percentages of rotting varied from 1.4% to 10.5% with significant differences (P > 0.05) between the isolates. The highest percentage of rot was caused by isolate Scr4 (10.5%) and the lowest, with isolate Scr9 (1.4%), which displayed no significant differences with isolates Scr3, Scr47 and Scr5 with percentages of 2.0%, 3.9% and 4.5% respectively, although there were such differences with the rest of the isolates (P > 0.05). Isolate Scr4 displayed no significant differences (P > 0.05) with isolates Scr7, Scr10, Scr17, Scr51 and Scr6 with percentages of 9.3%, 8.4%, 8.0%, 8.0% and 7.7%, respectively (Table 4). Symptoms and signs of the disease were observed, similar to those that occur in the field, which coincides with the results by Daami-Remadi et al. (2007) when they showed the pathogenicity of the fungus in var. Spunta potato.

Penetration of the pathogen and percentage of soft rot in potato tubers var. Fianna with artificial inoculation.
Penetration of the pathogen(mm) Soft rot (%)
Isolate N Meanx Isolate N Meanx
Control 10 0.0 a Control 10 0.0 a
Scr9 10 9.5 ab Scr9 10 1.4 ab
Scr3 10 10.0 ab Scr3 10 2.0 abc
Scr49 10 11.0 abc Scr47 10 3.9 abcd
Scr5 10 11.1 abc Scr5 10 4.5 abcde
Scr13 10 11.1 abc Scr13 10 4.5 bcde
Scr2 10 11.2 abcd Scr12 10 5.1 cdef
Scr12 10 11.5 bcde Scr8 10 5.3 defg
Scr8 10 12.2 cdef Scr49 10 5.6 defg
Scr14 10 12.3 def Scr2 10 6.0 efg
Scr50 10 12.4 def Scr48 10 6.1 efgh
Scr47 10 12.6 efg Scr14 10 6.2 fgh
Scr6 10 12.7 fg Scr50 10 6.2 fghi
Scr11 10 12.8 fg Scr11 10 7 ghij
Scr51 10 12.9 fg Scr54 10 6.9 ghij
Scr54 10 13.2 fgh Scr6 10 7.7 hijk
Scr10 10 13.6 ghi Scr51 10 8.0 hijk
Scr48 10 13.7 ghi Scr17 10 8.0 ijk
Scr17 10 14.0 ghi Scr10 10 8.4 jk
Scr7 10 15.2 hi Scr7 10 9.3 jk
Scr4 10 16.9 i Scr4 10 10.5 k

Means with at least a common letter are not significantly different (P > 0.05), Kruskal-Wallis. N= Repetitions

Symptoms of soft rot in Fianna variety potato tubers, caused by the isolate Scr17 of <italic>S. rolfsii</italic> by artificial inoculation (<bold>A</bold>) Tubers with symptoms of <italic>S. rolfsii</italic> in a moist chamber. (<bold>B and C</bold>) lesions with cottonlike mycelium caused by the fungus. (<bold>D</bold>) Transverse cut of potato tuber with soft rot.

The results of this work are relevant because new lines of investigations are opened in which may include studies on the pathogenicity of S. rolfsii in different agricultural crops, crop rotation and its impact on the sclerotia populations, as well as the use of Trichoderma species on the reduction of sclerotia production and its correlation with the reduction of the incidence of the disease in the field. This is because Trichoderma has been identified as a promising biological control agent against soil pathogens such as S. rolfsii in the potato crop (Rubayet and Bhuiyan, 2016; Chowdhury et al., 2023). The use of endemic Trichoderma species obtained from fields planted with potato where they have been exposed to the pressure of several fungicides, suggests that they have become resistant to these types of molecules. This implies that they could be used in combination with fungicides to which they are resistant, for the control of potato soft rot, as a strategy to reduce the doses of synthetic fungicides.

 Conclusions

A total of 239 commercial potato plots were sampled in Sonora and Sinaloa during the 2019-2020, 2020-2021 and 2021-2022 agricultural cycles. Out of these plots, 8.4% were positive for S. rolfsii, with a population density of 2 to 24 sclerotia per kg of soil. The identity of the fungus was confirmed using conventional techniques and molecular tools. The capacity of S. rolfsii to induce soft rot in Fianna variety potato tubers was confirmed, with a variation in the virulence between isolates. The penetration of the pathogen fluctuated between 9.5 and 16.9 mm, whereas the percentage of rot varied from 1.4 to 10.5%, with isolate Scr4 standing out as the most aggressive.

On the other hand, 26 Trichoderma spp. isolates were obtained, accounting for 10.9% of the plots in which the fungus was located, with a population density of 2 to 8 UFC g-1 of soil. Morphological and molecular characterization enabled the identification of T. asperellum, T. asperelloides, T. afroharzianum and T. azevedoi, which belong to the T. viride and T. harzianum complexes.

The findings of this work have significant implications for the management of soft rot in potato tubers. The identification of native Trichoderma species with antagonistic potential opens future lines of investigation related to the implementation of biological control strategies, particularly in systems in which resistance to fungicides is a limitation. In addition, the variability in the virulence of S. rolfsii suggests the need for additional studies to understand their interaction with the host and the environment, as well as to develop cultural practices that reduce the survival of sclerotia in the soil.

 Limitations. This paper did not include <italic>Trichoderma</italic> and <italic>S. rolfsii</italic> confrontation tests.

Conflict of interest. None to be reported.

Funding. UAO and Local SV Board, Valle del Fuerte.

Acknowledgements. The authors would like to thank the potato farmers in Sinaloa and Sonora for their disposition towards this investigation.

Contribution of authors. The first four authors contributed with lab, analysis and interpretation work. The second author was responsible for the experimental conception, writing and revision of the manuscript. The fifth and sixth authors were in charge of field management sampling.

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