The Need and Opportunity to Update the Inventory of Plant Pathogenic Fungi and Oomycetes in Mexico.

Mexico generates specific phytosanitary regulations for each product and origin to prevent the entry of quarantine pests and/or delay their spread within the national territory, including fungi and oomycetes. Phytosanitary regulations are established based on available information on the presence or absence of these pathogens in the country; however, the compilation and precise analysis of reports is a challenging task due to many publications lacking scientific rigor in determining the presence of a taxon of phytosanitary interest in the country. This review evaluated various studies reporting the presence of plant pathogenic fungi and oomycetes in Mexico and concluded that some lists of diseases and phytopathogenic organisms lack technical-scientific basis. Thus, it highlights the need and presents an excellent opportunity to establish a National Collection of Fungal Cultures and a National Herbarium for obligate parasites, as well as to generate a National Database of Phytopathogenic Fungi and Oomycetes present in Mexico, supported by the combination of morphological, molecular, epidemiological, pathogenicity, symptom, and micrograph data. If realized, this would have a direct impact on many future applications related to various topics, including quarantines, risk analysis, biodiversity studies, and monitoring of fungicide resistance, among others.

First Report of Golovinomyces bolayi Causing Powdery Mildew on Lettuce in Mexico.

In August 2022, powdery mildew symptoms were detected on lettuce (Lactuca sativa) in a commercial field located in Quecholac, Puebla, Mexico. Signs appeared as whitish powdery masses on leaves. Disease incidence was about 100% and signs covered up to 40% of leaf surface. Mycelium was amphigenous forming white patches. Hyphal appressoria were indistinct or nipple-shaped and solitary. Conidiophores (n= 30) were hyaline, erect, arising from the upper surface of hyphal mother cells or lateral, and of 90 to 201 µm long. Foot cells were cylindrical, of 49 to 92 × 10-15 µm, followed by 1-3 shorter cells, and forming conidia in chains. Conidia (n= 100) were hyaline, ellipsoid-ovoid, doliiform-subcylindrical, 27 to 40 × 14 to 20 µm. Conidial germination belonging to the Euoidium type. Chasmothecia were not observed. The morphological characters were consistent with those of Golovinomyces bolayi (Braun et al. 2019). A voucher specimen was deposited in the Herbarium of the Department of Agricultural Parasitology at the Chapingo Autonomous University under accession number UACH451. To confirm the identification of the fungus, genomic DNA was extracted from conidia and mycelium following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region was amplified by PCR using the primers ITS5/ITS4 (White et al. 1990) and sequenced. The resulting 506 bp sequence had 100% identity to those of G. bolayi (LC417109 and LC417106). Phylogenetic analyses using the Maximum Likelihood and Maximum Parsimony methods were performed and confirmed the results obtained in the morphological analysis. The isolate UACH451 grouped in a clade with isolates of G. bolayi. The ITS sequence was deposited in GenBank under accession number OR467546. Pathogenicity was confirmed by gently dusting conidia onto ten leaves of healthy lettuce plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 30 ºC, and relative humidity of 70%. All inoculated leaves developed similar symptoms to the original observation after 10 days, whereas control leaves remained disease free. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed. Based on morphological data and phylogenetic analysis, the fungus was identified as G. bolayi. This pathogen has been previously reported causing powdery mildew on lettuce in Argentina, Canada, Chile, Ecuador, Peru, USA and Venezuela (Braun et al. 2019; Mieslerová et al. 2020). To our knowledge, this is the first report of G. bolayi causing powdery mildew on lettuce in Mexico.

Preventive and Curative Effects of Treatments to Manage Strawberry Root and Crown Rot Caused by Neopestalotiopsis rosae.

Strawberry root and crown rot caused by the fungus Neopestalotiopsis rosae is an emerging disease that has caused yield losses reaching 70% in Mexico and other regions worldwide. This research evaluated the effects of biological and chemical fungicides applied as preventive and curative applications for controlling root and crown rot caused by N. rosae in strawberries under greenhouse conditions. Treatments included these chemical fungicides: prochloraz, prochloraz+thiram, cyprodinil+fludioxonil, difenoconazole+azoxystrobin, iprodione, captan, thiram, pydiflumetofen+fludioxonil, fluxapyroxad+pyraclostrobin, and hymexazol; each applied at commercial doses. Also tested were biological treatments based on Trichoderma koningiopsis, Trichoderma asperellum, Streptomyces sp., and Bacillus amyloliquefaciens strain D747 (B. velezensis). Disease incidence, severity, plant mortality, root length, and dry weight were determined. Results showed that overall, preventive applications of the fungicides pydiflumetofen+fludioxonil, cyprodinil+fludioxonil, and prochloraz resulted in the smallest area under the disease progress curve, and lowest final disease incidence, severity, and plant mortality. An intermediate group of effective treatments entailed hymexazol, iprodione, T. asperellum, and T. koningiopsis (50-75% efficacy). Treatments with greater efficacy (99 to 100%), prochloraz pydiflumetofen+fludioxonil, cyprodinil+fludioxonil, and prochloraz, also had maximal total plant biomass vis-à-vis the untreated control. In contrast, each treatment's efficacy was significantly reduced when applied curatively (0 to 37% treatment efficacy). These results suggest that certain treatments are useful for controlling strawberry root and crown rot caused by N. rosae, when applied preventively (as root dipping). These results will contribute to design more effective management programs of root rot and crown rot caused by N. rosae on strawberry.

First Report of Peach Scab Caused by Cladosporium tenuissimum in Mexico.

Peach (Prunus persica) is one of the most popular stone fruits in the world. From 2019 to 2022, typical scab symptoms were observed on 70% of peach fruits in a commercial orchard in Tepeyahualco, Puebla, Mexico (19°30'38"N 97°30'57"W). Fruit symptoms are black circular lesions of 0.3 mm in diameter. The fungus was isolated from symptomatic fruit pieces that were surface sterilized with 1% sodium hypochlorite for 30 s, rinsed in autoclaved distilled water three times, placed on PDA medium, and incubated at 28°C in darkness for 9 days. Cladosporium-like colonies were isolated. Pure cultures were obtained by single spore culture. Colonies on PDA showed aerial mycelium abundant, smoke-grey, fluffy, and with margin glabrous to feathery. Conidiophores were solitary, long, intercalary conidia narrow erect, macro- and micronematous, straight or slightly flexuous, cylindrical-oblong, olivaceous-brown, and often subnodulose. Conidia (n= 50) catenate in branched chains, obovoid to limoniform, sometimes globose, aseptate, olivaceous-brown, apically rounded, 3.1 to 5.1 × 2.5 to 3.4 µm. Secondary ramoconidia (n= 50) were fusiform to cylindrical, smooth-walled, 0-1-septate, pale brown or pale olivaceous-brown, measuring 9.1 to 20.8 × 2.9 to 4.8 µm. Morphology was consistent to that described for Cladosporium tenuissimum (Bensch et al. 2012; 2018). A representative isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Department of Agricultural Parasitology at the Chapingo Autonomous University under the accession number UACH-Tepe2. To further confirm the morphological identification, total DNA was extracted using the cetyltrimethylammonium bromide method (Doyle and Doyle 1990). The internal transcribed spacer (ITS) region, partial sequences of the translation elongation factor 1-alpha (EF1-α) and actin (act) genes were amplified by PCR, and sequenced using the primer pairs ITS5/ITS4 (White et al. 1990), EF1-728F/986R, and ACT-512F/783R (Carbone and Kohn 1999), respectively. The sequences were deposited in GenBank under the accession numbers OL851529 (ITS), OM363733 (EF1-α), and OM363734 (act). BLASTn searches in GenBank showed 100% identity with available sequences of Cladosporium tenuissimum accession (ITS: MH810309; EF1-α: OL504967; act: MK314650). A phylogenetic analysis using the maximum likelihood method placed isolate UACH-Tepe2 in the same clade as C. tenuissimum. To verify the pathogenicity of the fungus, 20 healthy peach fruits were inoculated with four drops of 15 µl of a conidial suspension (1 × 106 spores /ml). Ten control fruit were treated with sterilized water. All the fruits were kept in a moist chamber at 25°C for 10 days. Circular and necrotic lesions were produced eight days after inoculation, whereas control fruits remained healthy. Pathogenicity test was conducted three times with similar results. Fungal colonies were reisolated from the artificially inoculated fruit, thus fulfilling Koch's postulates. Cladosporium tenuissimum has been previously reported to cause diseases on strawberry, cashew, papaya, and passionfruit in Brazil (Rosado et al. 2019; Santos et al. 2020), as well as diseases on pitaya, hydrangea, and carnation in China (Xu et al. 2020; Li et al. 2021; Xie et al. 2021). Cladosporium carpophilum is reported as the causal agent of peach scab. The environmental conditions for the development of C. carpophilum are 20-30 °C in warm humid areas (Lawrence and Zehr 1982), however, in this case the infection by C. tenuissinum occurred in a temperate semi-dry climate, with temperatures of 5 -15 °C and R.H. less than 50 % with an incidence of 80 %. To our knowledge, this is the first report of Cladosporium tenuissimum causing peach scab in Mexico and worldwide.

First Report of Puccinia menthae Causing Leaf Rust on Peppermint (Mentha x piperita) in Mexico.

Peppermint (Lamiaceae) is an aromatic herb with culinary, medicinal, and industrial properties. In June 2022, symptoms and signs of foliar rust were observed in four commercial fields of peppermint (Mentha × piperita) in San Buenaventura Tecalzingo, San Martín Texmelucan, Puebla, Mexico (19°14'34.0"N 98°27'25.4"W; 19°14'16.7"N 98°27'21.4"W; 19°14'37.0"N 98°27'07.7"W; 19°15'00.6"N 98°26'54.7"W). Two diseased plants were collected at each site. The disease was present in 50% of the plants and the damaged foliar tissue was under 17%. Initial symptoms included small chlorotic spots on the adaxial surface of the leaves, which later spread to form a necrotic area surrounded by a broad chlorotic halo. Necrosis developed only in the presence of abundant reddish-brown pustules on the abaxial surface of the leaf, while smaller pustules were observed on the adaxial surface. The signs were detected as numerous reddish-brown pustules on the abaxial surface of the leaves. The infected leaves of all samples showed subepidermal uredinia, erumpent, with hyaline and cylindrical paraphyses. Urediniospores (n = 50) were hyaline to light brown, echinulate, with two germinative pores, obovoid (16.5-26.5 × 11.5-25.5 µm, mean ± SD = 22 ± 1.6 × 19 ± 0.4 µm and 0.6 µm of wall thickness), individually supported on pedicels. Morphological characteristics aligned most closely with the description of Puccinia menthae by Kabaktepe et al. (2017) and Solano-Báez et al. (2022). A voucher specimen was deposited in the Herbarium of the Department of Plant-Insect Interactions at the Biotic Products Development Center of the National Polytechnic Institute under accession no. IPN 10.0115. From one sample, genomic DNA was extracted, and the 28S gene region of rDNA was amplified by a nested PCR using the primer sets Rust2inv (Aime, 2006) and LR6 (Vilgalys and Hester, 1990), and Rust28SF (Aime et al., 2018), and LR5 (Vilgalys and Hester, 1990) for the first and second reactions, respectively. The obtained sequence (GenBank accession No. OQ552847) showed 100% homology (902/1304bp) with the type-specimen sequence of P. menthae (DQ354513) from Cunila origanoides from USA (Aime, 2006). A phylogenetic analysis using Maximum Likelihood including a published 28S dataset for Puccinia species was executed and the isolate IPN 10.0115 was grouped into a clade of P. menthae with bootstrap support value of 100%. Pathogenicity was assessed by spraying a suspension of urediniospores (1×104 spores/ml) of the isolate IPN 10.0115 onto six healthy peppermint plants (Mentha × piperita) that were 30 days old, while six other plants were sprayed with sterile distilled water. All plants were kept in a wet chamber for 48 h at temperatures from 28±2°C and relative humidity of 95%, after which the plastic bag was removed. All inoculated plants developed disease symptoms after 15 days, whereas the control plants remained symptomless. The pathogenicity assay was conducted twice with similar results. The morphology of the pathogen recovered from the pustules of the inoculated plants was identical to that originally recollected, thus fulfilling Koch'postulates. To our knowledge, this is the first report of Puccinia menthae causing leaf rust on Mentha × piperita in Mexico. This species has been previously identified using morphological characteristics in Brazil, Canada, Poland, and USA on Mentha × piperita (Farr and Rossman, 2023). Since the disease defoliates peppermint plants reducing yield, further information on disease management is needed.

Occurrence and Distribution of Physiological Races of Exserohilum turcicum in Maize-Growing Regions of Mexico.

Turcicum leaf blight (TLB) is a common foliar disease of maize in Mexico that is caused by the fungal pathogen Exserohilum turcicum. The most effective management strategy against TLB is monogenic race-specific resistance. Among the 140 E. turcicum isolates from symptomatic leaves collected from maize fields in Mexico, 100 were obtained from tropical (Veracruz) and temperate areas (Estado de México) between 2010 and 2019, and 40 isolates were obtained from tropical (Sinaloa, Tamaulipas, Veracruz, and Chiapas), subtropical (Nayarit, Jalisco, and Guanajuato), and temperate areas (Estado de Mexico, Hidalgo, and Puebla) collected in 2019. All the isolates caused TLB symptoms on the positive control (ht4), showing that they were all pathogenic. Six physiological races of E. turcicum (2, 3, 23, 3N, 23N, and 123N) were identified based on resistant or susceptible responses displayed by five maize differential genotypes (A619Ht1, A619Ht2, A619Ht3, B68HtN, and A619ht4). The most common was race 23, accounting for 68% of the isolates, followed by races 23N, 123N, 3, 2, and 3N at 15, 8, 6, 2, and 1%, respectively. Race 123N was able to infect the greatest number of maize differential genotypes used in the study. Race 123N was detected in Sinaloa and Estado de México. Race 3 was detected in Nayarit and Jalisco. Race 2 was detected in Jalisco, Estado de México, and Veracruz, and race 3N was detected in Tamaulipas. Race 23 was equally dominant in the tropical, subtropical, and temperate regions, while race 123N was more common in the tropical environment, and race 23N was more common in the tropical and temperate environments. There was no evidence for shifts in the races between 2010 and 2019.

Efficacy of Biorational Products for Managing Diseases of Tomato in Greenhouse Production.

Gray mold (Botrytis cinerea), late blight (Phytophthora infestans), powdery mildew (Leveillula taurica), pith necrosis (Pseudomonas corrugata), and bacterial canker (Clavibacter michiganensis) are major diseases that affect tomato (Solanum lycopersicum L.) in greenhouse production in Mexico. Management of these diseases depends heavily on chemical control, with up to 24 fungicide applications required in a single season to control fungal diseases, thus ensuring a harvestable crop. While disease chemical control is a mainstay practice in the region, its frequent use increases the production costs, likelihood of pathogen-resistance development, and negative environmental impact. Due to this, there is a need for alternative practices that minimize such effects and increase profits for tomato growers. The aim of this study is to evaluate the effect of biorational products in the control of these diseases in greenhouse production. Four different treatments, including soil application of Bacillus spp. or B. subtilis and foliar application of Reynoutria sachalinensis, Melaleuca alternifolia, harpin αß proteins, or bee honey were evaluated and compared to a conventional foliar management program (control) in a commercial production greenhouse in Central Mexico in 2016 and 2017. Disease incidence was measured at periodic intervals for six months and used to calculate the area under the disease progress curve (AUDPC). Overall, the analysis of the AUDPC showed that all treatments were more effective than the conventional program in controlling most of the examined diseases. The tested products were effective in reducing the intensity of powdery mildew and gray mold, but not that of bacterial canker, late blight, and pith necrosis. Application of these products constitutes a disease management alternative that represents cost-saving to tomato growers of about 2500 U.S. dollars per production cycle ha-1, in addition to having less negative impact on the environment. The products tested in this study have the potential to be incorporated in an integrated program for management of the examined diseases in tomato in this region.

Occurrence of the Fusarium incarnatum-equiseti Species Complex Causing Fusarium Head Blight of Wheat in Mexico.

Fusarium head blight (FHB) is one of the most important diseases affecting wheat production worldwide. In Mexico, Fusarium boothii and F. avenaceum are the dominant species causing FHB of wheat (Cerón-Bustamante et al. 2018). During the 2017 to 2019 surveys, FHB symptoms were observed in wheat fields in the Highlands region of Mexico. Symptomatic spike samples were collected from 19 wheat fields in five states (Tlaxcala, Hidalgo, Puebla, Estado de México, and Morelos). Fusarium-like colonies were consistently isolated on potato dextrose agar (PDA) and 95 monoconidial isolates were obtained. Morphological features of seven isolates were consistent with the description of the Fusarium incarnatum-equiseti species complex (Xia et al. 2019). On PDA, colonies exhibited white and fluffy aerial mycelia, with diffused pink pigment on the reverse side after 7 days of incubation at 25℃. On carnation leaf agar (CLA), macroconidia (n = 100) were hyaline, falcate, with 3 to 6 septa, measuring 25.2 to 43.1 × 2.8 to 5.1 µm, and foot-shaped basal cell. Chlamydospores were ellipsoidal or subglobose and produced in chains. These seven isolates were selected for multilocus phylogenetic analysis and pathogenicity tests. Isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Department of Agricultural Parasitology at the Chapingo Autonomous University under acc. nos. UACH428 to UACH434. For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region, partial sequences of translation elongation factor 1-alpha (EF1-α) and the second largest subunit of RNA polymerase II (RPB2) genes were amplified, and sequenced with the primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and RBP2-5F/RPB2-7R (Liu et al. 1999), respectively. A phylogenetic tree, including published ITS, EF1-α, and RPB2 sequence data, was constructed for the Fusarium incarnatum-equiseti species complex (FIESC) based on Maximum Likelihood. Three species of the FIESC were identified into F. pernambucanum (five isolates), F. sulawesiense (one isolate), and F. clavum (one isolate). The sequences were deposited in GenBank with accession nos. OL347713 to OL347719 for ITS, OL365078 to OL365084 for EF1-α, and OL365072 to OL365077 for RPB2. The pathogenicity of the isolates was confirmed on wheat cv. Nana F2007 at the flowering stage in a glasshouse assay. The heads of 20 wheat plants were sprayed with a conidial suspension (1 × 105 spores/ml) of each isolate. Ten plants mock-inoculated with sterilized water served as the controls. All plants were placed in a moist chamber for 48 h. At 10 days after inoculation, typical FHB symptoms were visible on the inoculated plants, whereas the control plants remained asymptomatic. The pathogenicity test was repeated twice with similar results. The fungi were reisolated from the infected heads and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch's postulates. Previously, three isolates of Fusarium sp. belonging to the FIESC, were associated with FHB of wheat in Mexico (Cerón-Bustamante et al. 2018); however, this is the first report of F. pernambucanum, F. sulawesiense, and F. clavum causing FHB of wheat in Mexico and worldwide (Farr and Rossman 2021). Further studies should be focused on determining the distribution, prevalence, and toxigenic potential of the isolates of the FIESC associated with wheat diseases in Mexico.

First Report of Puccinia menthae Causing Leaf Rust on Spearmint (Mentha spicata) in Mexico.

Spearmint (Lamiaceae) is an aromatic herb widely cultivated in Mexico for its culinary, medicinal, and industrial properties. In May 2020, symptoms and signs of rust were observed in a two-ha commercial crop of spearmint in Cuautla, Morelos (18°50'26.6"N 98°57'31.9"W), Mexico. The disease incidence was 85% and the severity was 23%. Initial symptoms included chlorotic spots on the adaxial surface of the leaves. At advanced stages of the disease, necrotic spots surrounded by chlorotic halos were developed, and later the plants were defoliated. The signs were observed as numerous orange to reddish-brown erumpent pustules primarily on the abaxial surface of the leaves. Microscopic examination of the samples revealed the presence of subepidermal uredinia, erumpent, with hyaline and cylindrical paraphyses. Urediniospores (n = 50) were hyaline to light yellow, globose to obovoid, measuring 17-27  11-25 µm, including 0.6-0.7 µm wall thickness, individually supported on pedicels, echinulate, with two germinative pores. Morphological features of the fungus correspond with previous descriptions of Puccinia mentha by Kabaktepe et al. (2017). A voucher specimen (accession no. UACH448) was deposited in the Department of Agricultural Parasitology Herbarium at the Chapingo Autonomous University. To confirm identification, the 28S gene region of rDNA was amplified from one sample by a nested PCR using the primer sets Rust2inv (Aime, 2006) and LR6 (Vilgalys and Hester, 1990), and Rust28SF primers (Aime et al., 2018), and LR5 (Vilgalys and Hester, 1990) for the first and second reactions, respectively. The sequence of our specimen (GenBank accession No. OL878354) showed 100% homology (923/1304bp) with the type-specimen sequence of P. menthae (GenBank accession No. DQ354513) from Cunila origanoides from USA (Aime, 2006). Also, a phylogenetic analysis (Bayesian inference) including a published 28S dataset for Puccinia species was performed and the isolate UACH448 was grouped into a clade with P. menthae. Pathogenicity was demonstrated by spraying five ml of a suspension of urediniospores (1104 spores/ml) recovered from infected leaves onto leaves of ten healthy spearmint plants and ten noninoculated plants served as controls. All plants were maintained at temperatures from 28 to 35°C and relative humidity ranging from 70 to 80%. All inoculated plants developed the characteristic symptoms of the disease after 14 days, whereas the control plants remained symptomless. The pathogenicity test was performed twice with similar disease response. The morphological characteristics of the pathogen recovered from all the inoculated plants were identical to that originally inoculated, thus fulfilling Koch's postulates. To our knowledge, this is the first report of Puccinia menthae causing leaf rust on Mentha spicata in Mexico. This species has been previously identified in Australia (Edwards et al. 1999), New Zealand (Beresford et al. 1982), and USA (Farr and Rossman, 2021) on Mentha spicata. This disease reduces considerably the quality of peppermint plants, so it is necessary to develop management strategies.

Biological activity of Claviceps gigantea in juvenile New Zealand rabbits.

The Ascomycete fungus Claviceps gigantea infects maize kernels and synthetizes several alkaloids, mostly dihydrolysergamides. There is limited information on the damage these toxins cause in mammals, despite reports from infested areas with 90% presence of the fungus sclerotia. With this background, it was decided to determine the biological activity of chemical compounds present in sclerotia of C. gigantea in rabbits 38 days after weaning. Sclerotia of C. gigantea were collected in fields with high incidence of the disease, ground and analysed for nutrients. Experimental diets were prepared with four treatments, where sclerotial powder was added, substituting for alfalfa flour in increasing proportions [C. gigantea/alfalfa flour (0:100, 5:95, 15:85 and 25:75)]. Total ergot alkaloid content was analysed by high-performance liquid chromatography. Male juvenile rabbits were utilised and distributed in completely randomised design with four replications. Initial weight was recorded in each animal, and experimental diet was offered. In this study, weight of animals, feed consumption and feed conversion were evaluated in individual animals. Blood samples were taken for haemograms, and finally euthanasia was practiced. The consumption of C. gigantea had a negative effect on body weight and feed consumption. The necropsies showed anomalies proportional to the consumption of feed contaminated with the fungus.