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.

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.

Detection of Brasiliomyces malachrae Causing Powdery Mildew on Ornamental Cotton (Gossypium hirsutum) plants in Mexico.

Cotton (Gossypium L.; Malvaceae) is the most important fiber crop worldwide, also as a source of vegetable protein and edible oil. Cultivated species of cotton were apparently domesticated independently in four separate regions, in both the Old and the New World. Due to its economic importance, it is necessary to study the diseases that limit its production. During July of 2020-2022, symptoms of powdery mildew were observed on 80 ornamental cotton plants in a nursery located in Cuautla (18°52'38"N; 98°58'28"W), Morelos, Mexico. Disease incidence was 29%. Signs first appeared as small white colonies, which subsequently developed into abundant mycelial grown mainly on the upper leaf surface. White patches of mycelia were observed on leaves. In advanced stages of the disease, plants exhibited symptoms of yellowing, necrosis, and early defoliation. Microscopic analysis from 10 plant samples showed that mycelia were amphigenous, epiphyllous, in thin patches and evanescent. Hyphae were hyaline, thin walled and hyphal appressoria were simply lobed. Chasmothecia (n=50) were sub-aggregate, generally spherical to subglobose (46-61 µm in diameter), whitish, subhyaline, smooth, with a peridium of a single cell layer and appendages were absent. Three asci per chasmothecia, subspherical, 30-44 × 26-38 µm, with 4-6 ascospores per ascus. Ascospores were hyaline, ellipsoid to ovoid (16-23 × 10-18 µm). The asexual phase was not observed. The characteristics observed correspond to Brasiliomyces malachrae (Braun and Cook 2012; Cabrera et al. 2018). 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 10.0114. To confirm identification, DNA was recovered from the fungus and the internal transcribed spacer (ITS) from one sample was amplified by PCR, using the primers ITS1/ITS4 (White et al. 1990). The sequence was deposited in GenBank (OQ546720) and showed 100% sequence homology (647/1642bp) with the type sequence of B. malachrae (LC191217) from Malvastrum coromandelianum in Argentina (Cabrera et al. 2018). Pathogenicity was verified through inoculation by gently dusting conidia from infected leaves onto leaves of five healthy cotton plants. Five noninoculated plants served as controls. All plants were maintained in a greenhouse at temperatures from 28±2°C and relative humidity ranging from 80±5%. The experiment was performed twice. Inoculated plants developed powdery mildew symptoms after 14 days, whereas the control plants remained healthy. The fungus on the inoculated leaves was morphologically identical to that originally observed on diseased plants, thus fulfilling Koch's postulates. To our knowledge, this is the first report of Brasiliomyces malachrae causing powdery mildew on Gossypium hirsutum in Mexico and North America (Farr and Rossman 2023). Powdery mildew on G. hirsutum caused by B. malachrae has been previously identified in Venezuela by Hanlin and Tortolero (1984). This disease could be a primary source of inoculum of powdery mildew for commercial cotton plantations, derived from the free movement of ornamental plants.

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 Golovinomyces sonchicola Causing Powdery Mildew on Sonchus oleraceus in Mexico.

Sonchus oleraceus, common sow thistle, is native to Europe, Northern Africa, and Western Asia. This plant has become a common weed throughout the world. In Mexico, this weed has become widely naturalized by replacing indigenous plants and invading many agricultural areas. During the spring of 2018 and 2019, common sow thistle plants showing typical symptoms and signs of powdery mildew, were collected from agricultural fields in Ahome, Sinaloa, Mexico. As much as 30% of plants were diseased and 60 to 95% of the foliage was affected. Mycelium was conspicuous and white-gray, and on stems and both surfaces of leaves. Appressoria were nipple-shaped to crenulate. Conidiophores (n= 30) were hyaline, cylindrical, erect, and up to 150 µm long. Foot-cells (n= 30) were distinctly curved, 47 to 75 × 10 to 13 µm, slightly constricted, followed by 1-3 shorter cells and formed conidia in chains. Conidia (n= 100) were ellipsoid to doliiform to subcylindrical, 28 to 37 × 14 to 19 µm, lacked fibrosin bodies, and germinated from the apex. Chasmothecia were not observed. The morphological characters were consistent with those of the anamorphic state of Golovinomyces sonchicola (Braun and Cook 2012, Jakse et al. 2019). A voucher specimen (accession no. FAVF215) was deposited in the Herbarium of the Faculty of Agriculture of El Fuerte Valley at the Autonomous University of Sinaloa (Juan Jose Rios, Sinaloa, Mexico). To confirm the morphological identification, genomic DNA was extracted from mycelium and conidia, and the internal transcribed spacer (ITS) region and part of the 28S gene were amplified by PCR and sequenced. The ITS region of rDNA was amplified using the primers ITS5/ITS4 (White et al. 1990). For amplification of the 28S rRNA partial gene, a nested PCR was performed using the primer sets PM3 (Takamatsu and Kano 2001)/TW14 (Mori et al. 2000) and NL1/TW14 (Mori et al. 2000) for the first and second reactions, respectively. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods (Braun et al. 2019), including ITS and 28S sequences of isolates of Golovinomyces spp. were performed and confirmed the results obtained from the morphological analysis. Isolate FAVF215 grouped in a clade with the other isolates of G. sonchicola. The ITS and 28S sequences were deposited in GenBank under accession numbers MW425872 and MW442972, respectively. Pathogenicity was demonstrated by gently dusting conidia from infected leaves onto leaves of 20 healthy plants and covered with plastic bags for 24 h. Ten non-inoculated plants served as controls. All plants were maintained in a greenhouse at 25 to 35ºC. All inoculated plants developed similar symptoms to those observed in the field from natural infections after 12 days, whereas powdery mildew symptoms and signs were not observed on control plants. The morphology asexual structures of fungus on inoculated plants were identical to those on naturally infected plants, fulfilling Koch's postulates. Inoculation tests were repeated twice with identical results. Based on the morphological data and phylogenetic analysis, the fungus was identified as G. sonchicola. This fungus has been reported causing powdery mildew on S. oleraceus in Germany, The Netherlands, Slovenia, and The United Kingdom (Farr and Rossman 2021). To the best of our knowledge, this is the first report of G. sonchicola causing powdery mildew on S. oleraceus in Mexico. This powdery mildew pathogen may represent an option for the biological control of common sow thistle.

First Report of Powdery Mildew on Leucophyllum frutescens Caused by Podosphaera xanthii in Mexico.

Leucophyllum frutescens (Scrophulariaceae family), commonly known as Texas sage or cenizo, is an evergreen shrub native to southwestern United States and northern Mexico. This plant is commercially sold as a native, drought-tolerant ornamental. During the spring of 2019 and 2020, typical symptoms of powdery mildew were found on cenizo plants growing as ornamentals in urban areas in the municipality of Ahome, Sinaloa, Mexico. Disease incidence was 95% from a sampled population of 120 plants. Initial symptoms of powdery mildew developed as irregular white colonies on upper leaf surfaces which expanded as infections progressed. In severe infections, leaves became distorted, exhibiting premature defoliation. Microscopic examination showed nipple-shaped appressoria. Conidiophores (n= 30) were hyaline, cylindrical, erect, 89.4 to 134.2 µm long, and forming catenescent conidia. Foot-cells were cylindrical, 35.7 to 65.3 × 10.2 to 13.5 µm, followed by 1-3 shorter cells. Conidia (n= 100) were hyaline, ellipsoid to ovoid, 27.9 to 40.5 × 13.8 to 18.9 µm, containing distinct fibrosin bodies. Germ tubes were simple to forked and laterally produced from the middle of conidia. Chasmothecia were not found during the sampling period on the infected leaves. Based on morphological characteristics, the fungus was identified as Podosphaera xanthii (Braun and Cook 2012). A voucher specimen (accession no. FAVF219) was deposited in the Herbarium of the Faculty of Agronomy of El Fuerte Valley at the Autonomous University of Sinaloa (Juan Jose Rios, Sinaloa, Mexico). To further confirm the identification, total DNA was extracted, and the internal transcribed spacer (ITS) region was amplified by PCR using the primers ITS5/ITS4 (White et al. 1990) and sequenced. The resulting 503 bp sequence (GenBank accession no. MT624793) had 100% coverage and 100% identity to those of P. xanthii (MT568609-MT568611, MT472035, MT309699, MT250855, MT242593). A phylogenetic tree using the maximum parsimony (MP) and maximum likelihood (ML) methods and including published ITS sequences for Podosphaera species was obtained. Phylogenetic analyses revealed that ITS sequence from FAVF219 isolate was grouped into a clade with P. xanthii. Pathogenicity was demonstrated by gently dusting conidia from infected leaves onto 50 leaves of five healthy plants. Five non-inoculated plants served as controls. All plants were covered with polyethylene bags for 48 h to maintain high humidity and were maintained in a greenhouse at temperatures ranging from 20 to 35ºC. All inoculated plants developed similar symptoms to the original observations after 19 days, whereas no symptoms of powdery mildew were observed on control plants. The fungus present on the inoculated plants was morphologically identical to that originally observed on diseased plants, fulfilling Koch's postulates. This fungus has been reported infecting members of the Cucurbitaceae in Mexico (Félix-Gastélum et al. 2017; Farr and Rossman 2020). However, to our knowledge, this is the first report of P. xanthii causing powdery mildew on a member of Scrophulariaceae, specifically L. frutescens in Mexico and worldwide. Further studies for monitoring and control strategies of powdery mildew on Texas sage are required.

First Report of Colletotrichum jiangxiense Causing Avocado Anthracnose in Mexico.

Mexico is the largest avocado (Persea americana) producer and exporter in the world. In January of 2019, typical symptoms of fruit anthracnose were observed on approximately 90% of avocado trees in backyards localized in Leonardo Bravo municipality in Guerrero, Mexico. Lesions on avocado fruits were circular, necrotic, and sunken, whereas the mesocarp showed a soft rot with dark brown discoloration. To perform fungal isolation, small pieces from adjacent tissue to lesions of five symptomatic fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri dish containing potato dextrose agar (PDA). Plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently isolated and seven monoconidial isolates were obtained. An isolate was selected as a representative for morphological characterization, molecular analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero (Accession No. CSAEG-CJ19). After 8 days on PDA, the colonies were gray on the upper surface, and with orange conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with rounded ends, 14.4 to 18.5 × 4.5 to 6.2 µm. Based on morphological features, the isolate was tentatively identified in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), ß-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced with primers ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), Bt2A/Bt2B (Glass and Donaldson 1995), and GDF/GDR (Templeton et al. 1992), respectively. BLAST analysis of the obtained sequences of the ITS, ACT, TUB2, and GAPDH genes revealed 100%, 99.63%, 99.77% and 100% identity with those of isolate LF687 of C. jiangxiense in GenBank (Accession numbers KJ955201, KJ954471, KJ955348, and KJ954902). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, and GAPDH data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CSAEG-CJ19 as C. jiangxiense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accession numbers ITS:MT011397; ACT:MN968784, TUB2:MN968786, and GAPDH:MN968785). To conduct Koch's postulates, 20 healthy avocado fruits (cv. Hass) were wounded with a sterile toothpick (2 mm in depth) and a drop of 15 µl of conidial suspension (1 × 105 spores/mL) was placed on each wound. Ten control fruit were wounded and treated with sterilized water. All the fruits were kept in a moist plastic chamber at 25°C for 8 days. All inoculated fruits developed circular and necrotic lesions (12 to 18 mm in diameter), 5 days after inoculation, whereas control fruits remained healthy. The fungus was consistently re-isolated from the inoculated fruits. Previously, C. jiangxiense has been reported as a pathogen on Camellia sinensis and Citrus sinensis in China (Farr and Rossman 2020). To our knowledge, this is the first report of C. jiangxiense causing anthracnose on avocado worldwide. This study shown another species in the C. gloeosporioides complex associated with avocado diseases in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.

Characterization of Neopestalotiopsis Species Associated with Mango Grey Leaf Spot Disease in Sinaloa, Mexico.

Mango is one of the most popular and nutritious fruits in the world and Mexico is the world's largest exporter. There are many diseases that directly affect fruit yield and quality. During the period 2016-2017, leaves with grey leaf spots were collected from 28 commercial mango orchards distributed in two main production areas in Sinaloa State of Mexico, and 50 Neopestalotiopsis isolates were obtained. Fungal identification of 20 representative isolates was performed using morphological characterization and phylogenetic analysis based on the internal transcribed spacer (ITS) region of ribosomal DNA, part of the translation elongation factor 1-alpha (TEF) and the ß-tubulin (TUB) genes. Phylogenetic analysis indicated that the 20 isolates from this study formed four consistent groups, however, overall tree topologies do not consistently provide a stable and sufficient resolution. Therefore, even though morphological and phylogenetic separation is evident, these isolates were not assigned to any new taxa and were tentatively placed into four clades (clades A-D). Pathogenicity tests on detached mango leaves of cv. Kent showed that the 20 isolates that belong to the four Neopestalotiopsis clades from this study and induce lesions on mango leaves. This is the first report of species of Neopestalotiopsis causing mango grey leaf spot disease in Mexico.

First Report of Colletotrichum siamense and C. gloeosporioides Causing Anthracnose of Citrus spp. in Mexico.

Citrus anthracnose, caused by Colletotrichum spp., is a major disease in many citrus-growing regions of the world. During the spring of 2019, symptoms of petal necrosis and necrotic lesions on fruits were detected on Mexican lime (Citrus aurantifolia), sweet orange (Citrus sinensis), and grapefruit (Citrus paradisi) trees in three commercial orchards distributed in northern Sinaloa (El Fuerte and Ahome municipalities), Mexico. Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium from symptomatic petals and fruits, and 30 monoconidial isolates (10 per orchard) were obtained. Five isolates were selected as representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were designated as FAVF355-FAVF359 and were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agronomy of El Fuerte Valley at the Autonomous University of Sinaloa (Mexico). Colonies grown on PDA at 25ºC were cottony, dense, with grayish white aerial mycelium and with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, 13.7 to 18.8 × 4.3 to 5.8 µm, with both ends rounded. Based on morphological features, the five isolates were tentatively identified in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß-tubulin (TUB2) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference for species belonging to the C. gloeosporioides species complex was constructed. The multilocus phylogenetic analysis distinguished the isolates FAVF355-FAVF357 as C. gloeosporioides sensu stricto and the isolates FAVF358-FAVF359 as C. siamense. The sequences were deposited in GenBank (accession numbers ITS: MT850050-MT850054; ACT: MT834528-MT834532; GAPDH: MT855979-MT855982; TUB2: MT834533-MT834536). Pathogenicity of the five isolates was verified on healthy fruits of their original host species. Five fruits per isolate were inoculated using the colonized agar plug method. Fruits were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) removed from the margin of a 6-days-old culture were placed onto three wound sites in each fruit. Non-colonized agar plugs were placed on the wounds of 10 fruits used as the control. The fruits were kept in a moist chamber at 25°C for 8 days. The experiment was repeated twice. All inoculated fruits developed circular and necrotic lesions 6 days after inoculation, whereas the control fruits remained symptomless. The fungi were consistently re-isolated from the diseased fruits and were morphologically identical to that originally inoculated, fulfilling Koch´s postulates. To date, only C. gloeosporioides sensu lato and C. acutatum sensu lato has been associated with sweet orange and Mexican lime in Mexico (Farr and Rossman 2020), whereas C. gloeosporioides sensu stricto has been recently recorded in a different area (Iguala, Guerrero) of Mexico (Cruz-Lagunas et al. 2020). To our knowledge, this is the first report of C. gloeosporioides sensu stricto causing anthracnose on sweet orange, and of C. siamense on Mexican lime in Mexico, as well as C. gloeosporioides s. s. causing disease on grapefruit in Sinaloa, Mexico.