[Ecological and biological benefits of the cosmopolitan fungus Trichoderma spp. in agriculture: A perspective in the Mexican countryside]. / Beneficios ecológicos y biológicos del hongo cosmopolita Trichoderma spp. en la agricultura: una perspectiva en el campo mexicano.

There is currently an extensive record of scientific studies on the general characteristics of filamentous fungus Trichoderma spp., which demonstrates its wide range of interrelation in ecosystems and its fungal activity that benefits the agricultural sector and agroindustry, as well as its importance in the preservation and restoration of the soil microbiota. The success of the biological and ecological benefits of Trichoderma is due to its reproductive capacity, as well as its efficiency in the use of soil nutrients; the efficacy of the genus has been reported against a variety of phytopathogenic fungi, as well as the potential to synthesize and release enzymes (cellulases, xylanases, and chitinases) that have been implemented in agroindustrial bioprocesses. It has also been reported that various species of Trichoderma spp. can produce auxins and gibberellin-type growth regulators, reported as growth promoters of some agricultural crops; however, their most relevant fact is their ability to prevail at certain doses of 'agrotoxic' active ingredients and contribute studies regarding processes for obtaining biofuel and bioremediation of the agricultural soil. In this overview, a general description of the current and relevant studies of the different subspecies of Trichoderma and their contribution in agriculture is made, presenting results obtained in vitro, in greenhouses and in the field. This analysis will serve as a starting point for future research in Mexico, specifically on the genus Trichoderma and its benefits for the Mexican countryside.

First Report of Vascular Wilt on Peanut in Mexico Caused by Fusarium incarnatum.

During the fall of 2020, wilt symptoms were presented in a commercial peanut field (Arachis hypogaea L.), variety 'CHAMPS' in Buenavista de Benito Juárez, México (18.460501 N, 98.627100 W). A peanut field was scout 80 days after planting, and plants presenting symptoms of root and crown rot, vascular chlorosis, and tissue death, were sampled. Disease incidence was estimated at 55% of the field. Isolations of the pathogen were made from stem and root tissues. These samples were disinfected by immersion in 1% sodium hypochlorite (NaClO) for 3 min and immersion in 70% ethanol for 1 min with 3 rinses with sterile distilled water. Subsequently, 0.5 cm fragments were removed and placed on media of potato dextrose agar (PDA) and Rose Bengal. Petri dishes were incubated in complete darkness at 26 °C for 7 days. Abundant aerial white mycelium was observed, which turned tan to brown and showed a slightly orange color on the back of the plate. Finally, pure cultures were obtained by single sporing (Aslam et al. 2020). Colonies identified as Fusarium spp. (Leslie and Summerell, 2006) were sub cultured on PDA agar media and Spezieller Nährstoffarmer (Pérez-Vázquez et al. 2022) to observe microscopic characteristics of ten isolates. Colonies of a representative strain (MA-PET-03) produced hyaline septate hyphae, macroconidia dorsoventrally curved, tapering towards both ends of 51-57 × 4.6-5.4 µm (n = 80) with most having 7 septa. Microconidia were unicellular, nonseptate, hyaline, and ovoid, 12.4-20.6 × 3.6-4.1 µm (n = 80). Chlamydospores were abundant and globose 5-11 µm diam (n = 80), intercalary, and solitary in short chains (Figure 1). The observed microscopic characteristics correspond to the description of Fusarium incarnatum (Khoa et al. 2006; Leslie and Summerell, 2006; Xia et al. 2019). The molecular analyses were done with genomic DNA extracted as previously reported by Pérez et al. (2022). A region from the translation elongation factor gene was PCR amplified using EF688/EF1251 primers (Alves et al. 2008) and from the calmodulin (CMDA) gene, using CALDF1 and CALDR1 primers (Noel et al. 2022). The corresponding PCR products were purified with the Gen Elute™ PCR Clean-Up Kit from Sigma-Aldrich Co. (St. Louis Mo. USA) and sequenced at Macrogen Inc. (Seoul, South Korea). The phylogenetic analysis was inferred using the Bayesian Inference method with 1 million generations, final standard deviation was 0.008516. The nucleotide substitution model for Calmodulin (CMDA) was GTR + G and for TEF1 GTR + I + G. This analysis showed that strain MA-PET-03 shared 100% identity (Figure 2) with F. incarnatum ex-type strain CBS 132.73 (CMDA: MN170342; TEF1: JMN1704761) from Pointed gourd (Trichosanthes dioica) in Malawi Africa. The sequences of strain MA-PET-03 were deposited in GenBank (CMDA: OQ679820; TEF1: OQ679821). The pathogenicity tests were carried out with a total of 20 peanut plants, variety 'CHAMPS', 18 days after having been sown in groups of five seeds in 250 g plastic pots, containing a sterilized mixture of Peatmoss and Agrellite (1:1 v./v), with four repetitions. The seeds were inoculated by immersion in 20 mL of spore suspension (106 conidia/mL) isolated from F. incarnatum for 10 min. The plants were maintained in a greenhouse (70% relative humidity and 28 °C) until the appearance of disease symptoms of. Likewise, 10 control plants were inoculated with sterile water. The experiment was repeated twice. The symptoms developed 15 days after inoculation, the plants presented symptoms of chlorosis, wilting of leaves, stems, and roots, a manifestation similar to that observed in the field, while the control plants remained healthy. F. incarnatum was consistently reisolated from inoculated stems and roots and identified by the microscopic characteristics described above. Peanut leaf blight and wilt disease caused by F. incarnatum has been reported in India (Thirumalaisamy et al. 2019). This first report emphasizes that this phytopathogen is a new threat for peanut producers in Mexico, which is why our finding suggests the need to seek new strategies for its control.

Biological Control of Charcoal Rot in Peanut Crop through Strains of Trichoderma spp., in Puebla, Mexico.

Charcoal rot is an emerging disease for peanut crops caused by the fungus Macrophomina phaseolina. In Mexico, peanut crop represents an important productive activity for various rural areas; however, charcoal rot affects producers economically. The objectives of this research were: (a) to identify and morphologically characterize the strain "PUE 4.0" associated with charcoal rot of peanut crops from Buenavista de Benito Juárez, belonging to the municipality of Chietla in Puebla, Mexico; (b) determine the in vitro and in vivo antagonist activity of five Trichoderma species on M. phaseolina, and (c) determine the effect of the incidence of the disease on peanut production in the field. Vegetable tissue samples were collected from peanut crops in Puebla, Mexico with the presence of symptoms of charcoal rot at the stem and root level. The "PUE 4.0" strain presented 100% identity with M. phaseolina, the cause of charcoal rot in peanut crops from Buenavista de Benito Juárez. T. koningiopsis (T-K11) showed the highest development rate, the best growth speed, and the highest percentage of radial growth inhibition (PIRG) over M. phaseolina (71.11%) under in vitro conditions, in addition, T. koningiopsis (T-K11) showed higher production (1.60 ± 0.01 t/ha-1) and lower incidence of charcoal rot under field conditions. The lowest production with the highest incidence of the disease occurred in plants inoculated only with M. phaseolina (0.67 ± 0.01 t/ha-1) where elongated reddish-brown lesions were observed that covered 40% of the total surface of the main root.

First Report of Macrophomina phaseolina Causing Charcoal Rot of Peanut (Arachis hypogaea L.) in Mexico.

Peanut (Arachis hypogaea L.) is the third most important oilseed crop in the world. The cultivated area in Mexico is currently 52,046 ha with a production of 91,109 ton in 2018 (FAO, 2020). Puebla state ranks third in the national production with 9,313 ton (SIAP, 2020). In September 2019, typical symptoms of charcoal rot (Macrophomina phaseolina (Tassi) Goid.) were observed in about 50% of cultivar Virginia Champs peanuts, and it affecting 1.5 ha located in Chietla (18° 27' 39" N; 98° 37' 11" W), Puebla, Mexico. Diseased plants showed brown discoloration in stem and root rot, with chlorotic foliage, dark microsclerotia were observed on the stem and premature dying. To isolate the causal agent of these symptoms, 20 infected plants were recovered and processed in the laboratory. Ten pieces of stem and root tissue were selected from each plant, cut into small pieces 5-mm in length, superficially disinfested with 1% sodium hypochlorite for 3 min, followed by three rinses with sterile distilled water. Later, dried on sterile paper and placed on Petri plates containing potato dextrose agar (PDA) medium, which were kept at 28°C for 7 days (12 h light and 12 h dark). Four colonies were purified via hyphal tip culture, fungus was consistently isolated from the analyzed tissues; additional microcultures were prepared to observe phenotypic characteristics. Colonies showed dense growth, with a gray initial mycelium, becoming black after 7 days. Microesclerotia with spherical to oblong in shape were observed after 5 days on PDA, with a black coloration, measuring an average of 74 µm width × 110 µm length (n=40). Phylogenetic analysis was conducted by amplification and sequencing of the internal transcribed spacer (ITS) region with the ITS5 and ITS4 primers (White et al. 1990). The obtained sequences were deposited in GenBank database under accession numbers: MW585378, MW585379, MW585380, and MW585381 containing approximately 601 bp of the ITS1-5.8S-ITS2 region (complete sequence); they were 99% identical with the reference sequence of Macrophomina phaseolina (GenBank accession KF951698) isolated in Phaseolus vulgaris from Mexico. Based on the symptoms in the field, colony morphology, color, and shape of the microsclerotia, and molecular identification, the fungus was identified as M. phaseolina (Tassi) Goid. The pathogenicity test was performed on peanut plants cultivar Virginia Champs grown on plastic pots with an autoclaved peat/soil mixture under greenhouse conditions (70% relative humidity and 28°C). Fifty two-month-old peanut plants were inoculated using the toothpick method. The toothpicks were previously sterilized and then placed in Petri plates with each of the four colonies of M. phaseolina until colonization. Small wounds were made with those toothpicks in the roots, and a sterile toothpick was used in the control plants, the assays were performed twice. After three weeks, the inoculated plants exhibited symptoms of wilting chlorosis on the leaves and brown to dark brown discoloration of the vascular ring, while control plants remained healthy. M. phaseolina was re-isolated from symptomatic root tissues and identified by phylogenetic approach, fulfilling Koch's postulates. To date, this fungus affects at least 372 hosts globally causing yield losses. Although in Mexico this fungus has been documented in Glycine max, Ipomoea batatas, Phaseolus vulgaris, Physalis ixocarpa, Saccharum officinarum, Sesamum indicum, Solanum melongena, S. tuberosum, and Sorghum bicolor (Farr and Rossman 2021). However, there are no reports of M. phaseolina as a potential pathogen on peanut; therefore, according to our knowledge, this is the first report of this fungus affecting A. hypogaea in Mexico.

Endophytic Trichoderma Species Isolated from Persea americana and Cinnamomum verum Roots Reduce Symptoms Caused by Phytophthora cinnamomi in Avocado.

Avocado root rot caused by the oomycete Phytophthora cinnamomi is a severe disease that affects avocado production in Mexico and worldwide. The use of biological control agents such as Trichoderma species isolated from places where the disease is always present, represents an efficient alternative to reduce losses. Thus, the objective of this research was to evaluate the biocontrol ability of 10 endophytic Trichoderma spp. strains against P. cinnamomi tested both in vitro and in the greenhouse. The endophytic Trichoderma spp. were recovered from Persea americana and Cinnamomum verum roots, isolated and purified on potato-dextrose-agar medium. Ten strains were identified by phylogenetic reconstruction of the internal transcribed spacer region of rDNA sequences as T. asperellum (T-AS1, T-AS2, T-AS6, and T-AS7), T. harzianum (T-H3, T-H4, and T-H5), T. hamatum (T-A12), T. koningiopsis (T-K8 and T-K11), and P. cinnamomi (CPO-PCU). In vitro dual-culture assay, the percentage of inhibition of radial growth (PIRG) between Trichoderma spp. and P. cinnamomi strains was measured according to the Bell's scale. PIRG results indicated that T-AS2 reached the highest value of 78.32%, and T-H5 reached the lowest value of 38.66%. In the greenhouse, the infection was evaluated according to the percentage of disease incidence. Plants with the lowest incidence of dead by avocado root rot were those whose seedlings were inoculated with T-AS2 and T-AS7, resulting in only 5% death by root rot caused by P. cinnamomi. The disease incidence of seedlings with wilt symptoms and death decreased more than 50% in the presence of Trichoderma spp. Relying on the results, we conclude that T. asperellum and T. harzianum contribute to the biocontrol of soil-borne pathogenic oomycete P. cinnamomi.