Persistent poverty and incidence-based melanoma mortality in Texas.

PURPOSE: Previous studies have shown that individuals living in areas with persistent poverty (PP) experience worse cancer outcomes compared to those living in areas with transient or no persistent poverty (nPP). The association between PP and melanoma outcomes remains unexplored. We hypothesized that melanoma patients living in PP counties (defined as counties with ≥ 20% of residents living at or below the federal poverty level for the past two decennial censuses) would exhibit higher rates of incidence-based melanoma mortality (IMM). METHODS: We used Texas Cancer Registry data to identify the patients diagnosed with invasive melanoma or melanoma in situ (stages 0 through 4) between 2000 and 2018 (n = 82,458). Each patient's PP status was determined by their county of residence at the time of diagnosis. RESULTS: After adjusting for demographic variables, logistic regression analyses revealed that melanoma patients in PP counties had statistically significant higher IMM compared to those in nPP counties (17.4% versus 11.3%) with an adjusted odds ratio of 1.35 (95% CI 1.25-1.47). CONCLUSION: These findings highlight the relationship between persistent poverty and incidence-based melanoma mortality rates, revealing that melanoma patients residing in counties with persistent poverty have higher melanoma-specific mortality compared to those residing in counties with transient or no poverty. This study further emphasizes the importance of considering area-specific socioeconomic characteristics when implementing place-based interventions to facilitate early melanoma diagnosis and improve melanoma treatment outcomes.

First Report of Colletotrichum tropicale Causing Anthracnose on Pitahaya Fruit in Mexico.

Pitahaya (Hylocereus spp.), also called dragon fruit, is a cultivated cactus that is native to Mexico as well as Central and South America. In October 2021, anthracnose symptoms were observed on fruit of pitahaya (Hylocereus costaricensis) in a commercial orchard located in Culiacán, Sinaloa, Mexico. Lesions on fruit were circular, sunken, dark brown and with halo. To fungal isolation, small pieces from adjacent tissue to lesions of symptomatic fruits were surface disinfested by immersion in a 2% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently observed on PDA and five monoconidial isolates were obtained. An isolate was selected as a representative for morphological identification, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited as CCLF186 in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa). On PDA, initially white colonies turned grey with abundant orange conidia masses at 8 days after incubation at 25 ºC. Conidia were cylindrical, with ends rounded, aseptate, hyaline, and measuring 15.2 to 18.9 × 4.3 to 6.4 µm (n= 100). Appressoria were terminal, subglobose to clavate, of 7.4 to 11.6 × 5.9 to 8.2 µm (n= 30). Setae were not observed. These morphological characters were consistent with those reported for the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To determine the phylogenetic identity of the isolate CCLF186, genomic DNA was extracted following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region, the ApMat intergenic region, as well as partial sequences of actin (act) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes were amplified and sequenced using the primers pairs ITS5/ITS4 (White et al. 1990), AM-F/AM-R (Silva et al. 2012), GDF/GDR, and ACT-512F/ACT-783R (Weir et al. 2012), respectively. The sequences were deposited in GenBank under accession nos. OP269659 (ITS), OP302778 (gapdh), OP302777 (act), and OP302779 (ApMat). BLASTn searches revealed high identity with sequences of C. tropicale (CBS 124949) for ITS (100%), ApMat (100%), act (100%), and gapdh (100%). A phylogenetic tree based on Bayesian inference and Maximum Likelihood methods, including published ITS, ApMat, act, and gapdh sequence datasets for isolates in the Colletotrichum gloeosporioides species complex was generated. The phylogenetic analysis based on the concatenated sequences clustered the isolate CCLF186 with the C. tropicale reference isolates. Pathogenicity of the isolate CCLF186 was confirmed on 10 healthy pitahaya fruits without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on two locations on each fruit. Ten control fruits were treated with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated pitahaya fruits exhibited sunken and necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation. Recently, C. tropicale causing anthracnose in dragon fruit (Selenicereus monacanthus) was reported from Philippines (Evallo et al. 2022). Now, this is the first report of C. tropicale causing fruit anthracnose in H. costaricensis in Mexico and worldwide. These findings provide a basis for research about the distribution and effective disease-management strategies.

First Report of Colletotrichum chrysophilum Causing Anthracnose on Nance (Byrsonima crassifolia) in Mexico.

Nance fruit [Byrsonima crassifolia (L.) HBK] is a native crop widely distributed in Mexico and South America (Medina-Torres et al. 2018). It has been reported that nance is a good source of active compounds with anti-inflammatory, neuropharmacological and antioxidant effects. In 2022, the annual production of nance fruit in Mexico was of 7,713.13 tons and average yield of 5.64 t/ha with economic value of 51,952.66 million pesos (SIAP, 2022). This production generated significant economic income for the communities at a local, regional, and national level. In January 2023, irregular necrotic spots were observed on leaves and fruit of nance in an orchard of 50 nance trees located in San Sebastián Nopalera (16°54'52.73"N; 97°47'50.35"W), Oaxaca, Mexico. The incidence of the disease ranged from 50 to 60% of the trees. Infected fruit first showed dark-brown lesions with defined borders that coalesced to form large necrotic area. Isolates were purified by single spore isolation method (Choi et al. 1999).Colletotrichum strains were grown in PDA medium and five monoconidial isolates were obtained. A representative sample was selected (CNC-NP3) and deposited in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. Colony on PDA was white with sparse aerial mycelium, and the center was dark grey with abundant acervuli. Conidia (n = 100) were hyaline, aseptate, cylindrical with rounded apex, 13.5 to 15.2 × 4.3 to 5.1 µm. Appressoria (n = 20) were terminal or lateral, obovoid to clavate and some with slightly lobed, 9.9 to 11.6 × 5.3 to 6.6 µm. Based on the morphology, the isolate was identified as belonging to the Colletotrichum gloeosporioides species complex (Jayawardena et al. 2016). The representative isolate CNC-NP3 was identified by multilocus phylogenetic analysis using nucleotide sequences of internal transcribed spacer (ITS), actin (ACT), ß-tubulin (TUB2), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (Jayawardena et al. 2016). The sequences were deposited in GenBank (accessions nos. OQ861102 (ITS), OQ870548 (ACT), OQ870549 (TUB2), OQ870550 (GAPDH). The phylogenetic analysis was carried out by Maximum likelihood method using concatenated sequences of ITS, ACT, TUB2 and GAPDH genes (Kozlov et al. 2019). The multilocus phylogenetic analysis revelated clearly the isolate CNC-NP3 as Colletotrichum chrysophilum. To confirm pathogenicity of CNC-NP3, 30 healthy fruits were inoculated. Fifteen disinfected nance with wounds and fifteen nance without wounds were inoculate with 10 µL of conidial suspension (1×105 spores/mL) from 7-day old culture. And controls were inoculated using sterile distilled water. Fruits were placed in a moist chamber covered with plastic bag at 25 °C for 48 h to maintain high humidity. After 4 days the inoculation sites development symptoms that were identical to those initially observed in the field, whereas the control group remained symptomless. The pathogenicity test was performed twice, with the same results. The pathogen was re-isolated from the lesion to fulfill Koch's postulates. Currently, Colletotrichum chrysophilum has been reported causing anthracnose disease in several crops: apple in New York (Khodadadi et al. 2020), papaya in Mexico (Pacheco- Esteva et al.2022), Blueberry (Soares et al. 2022) and banana in Brazil (Astolfi et al. 2022). To our knowledge, this is the first report of anthracnose in Byrsonima crassifolia caused by Colletotrichum chrysophilum in Oaxaca, 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.

First Report of Sclerotium rolfsii Causing Collar Rot of Guar (Cyamopsis tetragonoloba) in Mexico.

Cyamopsis tetragonoloba (Fabaceae), also known as guar or cluster bean, is an annual legume grown mainly for industrial purposes and also as an ingredient for animal feed. In October 2021, collar rot symptoms were observed in five guar fields located in Guasave, Sinaloa, Mexico. Abundant white mycelium, and later brown and small sclerotia were observed at the base of the stems. Diseased plants showed reduced growth, wilting, and drying of the entire plant. Disease incidence ranged from 15 to 40%. Samples were collected from each field at two phenological stages (vegetative and reproductive). For fungal isolation, symptomatic stems pieces were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed in sterilized distilled water two times, placed on PDA medium and incubated at 28°C in darkness for 3 days. Sclerotium-like colonies were consistently obtained and five isolates from five different fields were purified by the hyphal-tip method. Fungal colonies were white, cottony, and often forming fans. Sclerotia (1 to 2 mm diameter) were white at first and then gradually turned dark brown. Microscopic examination showed septate hyphae with some cells having clamp connections. A representative isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of Fuerte Valley at the Sinaloa Autonomous University under Accession no. FAVF647. For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region was amplified by PCR and sequenced using the primer pair ITS5/ITS4 (White et al. 1990). The sequence was deposited in GenBank (accession no. OM510466). BLASTn searches in GenBank showed 99.21 to 100% identity with the available sequences of Sclerotium rolfsii (accession nos. MK926446, MH854711, and KY175225). A phylogenetic analysis using the maximum Likelihood method placed isolate FAVF647 in the same clade as S. rolfsii. Pathogenicity tests were performed by inoculating 10 healthy guar seedlings (15-day-old) grown in pots. Four sclerotia were directly placed on the stem base of each plant. Five uninoculated guar seedlings were used as control. All plants were placed in a moist chamber at 25°C with a 12-h photoperiod for 2 days. Collar rot symptoms appeared on inoculated plants after 3 days, whereas control plants remained symptomless. Pathogenicity test was performed twice with similar results. The fungus was reisolated from the artificially inoculated plants, thus fulfilling Koch's postulates. Sclerotium rolfsii has been reported on guar plants in Australia, Brazil, Fiji, India, and the United States (Farr and Rossman 2022). To our knowledge, this is the first report of Sclerotium rolfsii causing collar rot of guar in Mexico. The disease is very common in guar fields in Sinaloa, Mexico, therefore additional studies are needed to develop effective disease-management strategies.

First Report of Leaf Blight Caused by Colletotrichum karsti on Peppermint (Mentha x piperitavar. citrata) in Mexico.

Spearmint (Mentha x piperita var. citrata (Ehrh.) Briq.: Lamiaceae) is an aromatic herb widely cultivated owing to its industrial properties. In June 2020, symptoms of leaf blight were observed on 1,500 peppermint plants in a commercial nursery located in Cuautla (18°52'18"N 98°57'58"W), Morelos, Mexico. The incidence of the disease was 89%. Symptoms were initially observed as irregular, small black necrotic spots, that grew rapidly until the leaves were blighted. Fungal isolation was done using diseased leaf tissue on potato dextrose agar (PDA) as described by Ayvar-Serna et al. (2020) and Colletotrichum-like colonies were obtained. Six isolates were purified by single spore culture and only a single morphotype was obtained. One isolate was used for pathogenicity tests, morphological characterization, and multilocus phylogenetic analysis. The isolate (accession no. UACH449) was deposited in the Culture Collection of Phytopathogenic Fungi of the Department of Agricultural Parasitology at the Chapingo Autonomous University. Colonies in PDA grow at a rate of 7.0-10.0 mm/d. After 14 days, the colony was white to orange, and conidia (n =100) were hyaline, cylindrical, and straight with rounded ends, measuring 15.0-17.0 × 4.5-6.5 µm. Appressoria were brown and bullet-shaped. In 28-day-old colonies, the formation of perithecia was observed. Asci were hyaline, unitunicate, 8-spored, fasciculate, and cylindrical to clavate. Ascospores (n =100) were hyaline, unicellular, allantoid, inaequilateral, often straight on the inner side, apices rounded, arranged biseriately within the asci, and measured 14-19 × 4.0-7.5 µm. Morphological features of the isolate placed it tentatively within the Colletotrichm boninense species complex (Damm et al. 2012). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), partial sequences of calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) (Damm et al. 2012) genes were amplified and sequenced. A phylogenetic tree including published ITS, CAL, GAPDH, and ACT data for Colletotrichum species was constructed and the isolate UACH449 was grouped in the clade of Colletotrichum karsti. Sequences were deposited in GenBank under the accession numbers: ITS, OL825605; CAL, OL855890; GAPDH, OL855891 and ACT, OL855889. Pathogenicity was tested by spraying a suspension of 1 × 10^5 conidia/ml, onto eight healthy peppermint plants 30-days-old var. citrata, while eight control plants were sprayed using sterile distilled water. All plants were kept at 25 +/- 2°C and 70% RH. The characteristic symptoms of the disease were observed seven days after inoculation, while control plants remained symptomless. The pathogenicity test was repeated twice. The fungus was consistently reisolated from the eight inoculated plants and was morphologically identical to that originally isolated from diseased leaves, fulfilling Koch's postulates. To date, this pathogen has not been reported on peppermint (Farr and Rossman, 2022). To our knowledge, this is the first report of Colletotrichum karsti causing foliar blight on peppermint worldwide. According to our field observations, this disease is a threat to the production of peppermint plants.

First Report of Soft Rot of Pitahaya Fruit Caused by Gilbertella persicaria in Mexico.

Pitahaya (Hylocereus spp.), also called dragon fruit, is a cultivated cactus that is native to Mexico as well as Central and South America. In September 2021, soft rot of fruit of H. ocamponis, H. undatus, and H. costaricensis was observed in a commercial orchard located in La Cruz de Elota, Sinaloa, Mexico. The disease occurred on approximately 15% of pitahaya fruit. Lesions on fruits were water-soaked and light brown, extending to the whole fruit and covered with mycelia, sporangiophores, and sporangia. Colonies of a fungus were consistently isolated on PDA medium and 10 isolates were obtained. Three isolates were selected and deposited in the Culture Collection of Phytopathogenic Fungi at the Research Center for Food and Development (Culiacán, Sinaloa) under accession nos. CCLF171-CCLF173. Colonies on PDA medium were initially white and later grayish. Sporangiophores were hyaline to light brown, and aseptate. Sporangia (n= 30) were initially light brown but became black at maturity, globose to subglobose, single, terminal, 65.8 to 117.2 µm in diameter, and longitudinally separated into two halves. Columellae (n= 20) were light brown, obovoid, 33.5 to 72.9 × 31.5 to 69.8 µm, with a distinct basal collar. Sporangiospores (n= 100) were hyaline, globose to ellipsoid, aseptate, 6.9 to 12.8 × 5.1 to 10.9 µm, with polar appendages. Chlamydospores were solitary or in chains, oval or irregular. Zygospores were not observed. Based on the morphological characters, the fungal isolates were identified as Gilbertella persicaria (Benny 1991). To confirm the identity, 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 ITS sequences were deposited in GenBank under the accession nos. OM301904-OM301906. A BLASTn search of these sequences showed 99.47 to 99.81% identity with the sequence MK301174 of G. persicaria from Hylocereus sp. in Taiwan. A phylogenetic analysis based on Maximum Likelihood method grouped the isolates CCLF171-CCLF173 within the G. persicaria clade. Pathogenicity of the three isolates was verified on healthy Hylocereus spp. fruit. Fruit of H. ocamponis, H. undatus, and H. costaricensis were surface sterilized with 80% ethanol, and dried. For each fungal isolate, five detached fruits were superficially wounded with a sterile toothpick and inoculated by placing 15 µL of a spore suspension (1 × 105 sporangiospores/mL). Sterile distilled water was applied to five healthy pitahaya fruits to serve as controls. All fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 6 days. All inoculated fruits developed rot 3 days after inoculation, whereas no symptoms were observed on the control fruits. The experiment was repeated twice with similar results. The fungi were consistently re-isolated from the diseased fruits, fulfilling Koch´s postulates. Gilbertella persicaria has been previously reported to cause stem rot, fruit rot, and wet rot in pitahaya (Hylocereus spp.) in Japan (Taba et al. 2011), China (Guo et al. 2012), and Taiwan (Lin et al. 2014), respectively. To our knowledge, this is the first report of G. persicaria causing soft rot of Hylocereus spp. fruit in Mexico. Additional studies are needed to develop effective disease-management strategies.

Occurrence of Curvularia pisi and C. muehlenbeckiae Causing Leaf Spot on Guar (Cyamopsis tetragonoloba) in Mexico.

Cyamopsis tetragonoloba (Fabaceae family), known as guar or clusterbean, is a drought-tolerant annual legume cultivated on a commercial scale focused on industrial gum production. In September 2021, symptoms of leaf spot were observed on guar plants in several commercial fields located at Guasave, Sinaloa, Mexico. Symptoms included round to oval, light brown lesions with dark margins. The disease incidence was estimated to be up to 30% in five fields. Curvularia-like colonies were consistently isolated, and 12 monoconidial isolates were obtained. Two representative isolates were selected to use downstream and were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of Fuerte Valley at the Sinaloa Autonomous University under Accession FAVF643 and FAVF645. On potato dextrose agar (PDA), colonies of both isolates FAVF643 (pale brown margin) and FAVF645 (lobate edge) were dark brown. Conidiophores of both isolates FAVF643 (paler towards apex and 76 to 191 × 3.5 to 5.2 µm) and FAVF645 (80 to 260 × 3.9 to 5.1 µm) were mostly straight, pale brown to dark brown, septate, and simple to branched. Conidia of both isolates FAVF643 (19.9 to 33.3 × 8.8 to 13.5 µm) and FAVF645 (18.5 to 27.1 × 9.1 to 13.1 µm) were curved, rarely straight, brown, with apical and basal cells paler than middle cells being pale brown, and 3-distoseptate. Morphology of both isolates FAVF643 and FAVF645 was consistent with that described for Curvularia (Marin-Felix et al. 2017; 2020). For phylogenetic identification, total DNA was extracted and PCR products sequenced from ITS5/ITS4 primers -the internal transcribed spacer (ITS) region (White et al. 1990) and GPD1/GPD2 - partial sequences of glyceraldehyde-3-phosphate dehydrogenase (gpdh) gene amplification. A phylogenetic tree based on Maximum likelihood including published ITS and gpdh for Curvularia spp. was constructed. Phylogenetic analyses showed that isolate FAVF643 grouped with the type strain C. pisi (CBS190.48) sequence, and the isolate FAVF645 grouped with the type strain C.muehlenbeckiae (CBS144.63) sequence. The resulting sequences were deposited in GenBank as: C. pisi OM802153 (ITS); OM835758 (gpdh), and C. muehlenbeckiae OM802154 (ITS); OM835759 (gpdh). The pathogenicity was verified on healthy guar plants. For each isolate, five plants were inoculated by spraying a conidial suspension (1 × 106 spores/ml) onto leaves until runoff. Five plants sprayed with sterile distilled water served as controls. All plants were kept in a moist chamber for two days, and subsequently transferred to a greenhouse for 12 days at temperatures ranging from 26 to 32°C. All inoculated leaves exhibited necrotic lesions with a dark margin 10 days after inoculation, whereas control plants remained symptomless. The fungi were consistently re-isolated from the diseased leaves and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch´s postulates. Curvularia lunata had been reported as the causal agent of leaf spot on guar in India (Chand and Verma 1968); however, to our knowledge, this is the first report of C. pisi and C. muehlenbeckiae causing leaf spot on guar in Mexico and worldwide.

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 Fusarium citri Causing Postharvest Fruit Rot of Chayote in Mexico.

In November 2018, symptoms of brown rot were observed on chayote (Sechium edule) var. nigrum spinosum with a 20% disease incidence of 120 harvested fruits in the National Germplasm Bank of Sechium edule, located in the Centro Regional Universitario Oriente (CRUO) from the Chapingo Autonomous University (Huatusco, Veracruz, Mexico). For fungal isolation, pieces from symptomatic fruits were surface disinfected by immersion in a 1.5% NaClO solution for 2 min, rinsed in sterile distilled water, placed in Petri plates containing potato dextrose agar (PDA) amended with kanamycin sulfate, and incubated at 25ºC. Fusarium-like colonies were consistently isolated on PDA and five monoconidial isolates were obtained. A representative isolate was selected for morphological characterization, phylogenetic analysis, and pathogenicity tests. On PDA, colonies exhibited white and fluffy aerial mycelia, with diffused pale brown pigment in the center at 7 days of incubation at 25℃ in darkness. Macroconidia (n= 100) were hyaline, falcate, with 4 to 5 septa, measuring 23.9 to 31.9 × 2.9 to 4.2 µm, and foot-shaped basal cells. Microconidia and chlamydospores were absent. Morphological features were consistent with the description of the Fusarium incarnatum-equiseti species complex (Xia et al. 2019). The isolate was deposited as FUS2 in the Culture Collection of Phytopathogenic Fungi of the Laboratory of Plant Pathology at the Colegio de Postgraduados. 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. DNA sequences were edited in BioEdit 7.2 and compared with those in the NCBI nucleotide database. Alignments were implemented in MEGA X using reference sequences from Fusarium spp. 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. The sequences were deposited in GenBank (accession nos. ON878083, ON890421, and ON890420). The phylogenetic analysis grouped the isolate FUS2 within the F. citri clade. Pathogenicity of the fungus was verified on 10 healthy chayote fruits var. nigrum spinosum previously disinfested by immersion in a 1% NaClO solution for 3 min and washed in sterile water. A total of 5 mL of a conidial suspension (1 × 106 spores/ml) was sprayed on each whole fruit. Ten control fruit were sprayed with sterile distilled water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 30 days. All inoculated fruits developed water-soaked brown lesions (3 to 5 cm in diameter) covered with white mycelium at 15 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation, fulfilling Koch´s postulates. Fusarium citri has been associated with Capsicum sp. and mandarin orange in China, Triticum sp. in Iran, alfalfa in Denmark, and lettuce in the Czech Republic and Italy (Farr and Rossman 2022). To our knowledge, this is the first report of F. citri causing postharvest fruit rot of chayote in Mexico and worldwide.

Sorghum Sheath Blight Caused by Fusarium spp. in Sinaloa, Mexico.

Sorghum (Sorghum bicolor) leaf sheath blight was observed for the first time in Sinaloa, Mexico in the summer of 2020. Fungal isolates were obtained from symptomatic tissue in potato dextrose agar. Fusarium spp. were associated with symptomatic plants in 10 sampling sites under field conditions. No root and stalk rot was observed during the sampling period. Analysis of fragments of the translation elongation factor alpha and RNA polymerase II second largest subunit genes indicated that all isolates belong to the Fusarium fujikuroi species complex (FFSC). Five groups were delineated from this complex: F. thapsinum, F. verticillioides, Fusarium sp. (four isolates), Fusarium sp. 4 (Fus4), and Fusarium sp. (Fus16), which is closely related to Fusarium madaense. The morphological characteristics (colony color and morphometry of conidia) of isolates with sequence similarities to those of F. thapsinum and F. verticillioides were in the expected range for these species. The morphology of isolates Fus7a, Fus7b, Fus11, and Fus17, as well as Fus4 and Fus16, were similar to those of the FFSC, specially to F. andiyazi and F. madaense. Inoculations of sorghum with representative isolates of F. thapsinum, F. verticillioides and the unidentified Fusarium species resulted in reddish brown lesions similar to those observed under field conditions; the original isolates inoculated were reisolated fulfilling the Koch's postulates. Although leaf sheaths on sorghum plants were heavily damaged, root and stalk rot were not observed in the greenhouse inoculations or under field conditions. Future research should focus on determining the identity of the unknown Fusarium spp. to design control measures for the disease. This is the first report of Fusarium spp. causing sorghum leaf sheath blight in Mexico.

First Report of Colletotrichum truncatum Causing Anthracnose of Guar (Cyamopsis tetragonoloba) in Mexico.

Guar (Cyamopsis tetragonoloba), is an annual legume belonging to the Fabaceae family and it is grown mainly for industrial purposes and also as an ingredient for animal feed. In September 2021, anthracnose symptoms were observed on guar fields distributed in Guasave, Sinaloa, Mexico. Disease incidence was estimated up to 15%. Diseased plants exhibited symptoms on leaves and pods. On leaves, lesions were irregular, necrotic, and often surrounded by a dark brown halo. On pods, necrotic and sunken lesions were developed. Colletotrichum-like colonies were consistently isolated on PDA medium and five monoconidial isolates were obtained. One isolate was selected as representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of Fuerte Valley at the Sinaloa Autonomous University under the accession number FAVF642. Colony on PDA was flat with an entire margin, dense, initially grayish white, then became dark gray with black microsclerotia and setae. Conidia (n= 50) were curved, hyaline, aseptate, with granular content, and measuring 20.4 to 25.8 × 2.8 to 3.9 µm. Setae were dark brown, straight, and septate. Morphological features matched those of Colletotrichum truncatum (Damm et al. 2009). For morphological identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR (Weir et al. 2012), and sequenced. The resulting sequences were deposited in GenBank under the accession nos. OM616022 (ITS), OM630461 (ACT), and OM630462 (GAPDH). BLASTn searches in GenBank showed 100%, 99.49%, and 99.15% identity to MT583079 (ITS), MG198003 (ACT), and MG703491 (GAPDH) of C. truncatum, respectively. A phylogenetic tree based on maximum Likelihood method and including published ITS, ACT, and GAPDH sequence data for Colletotrichum truncatum species complex was generated (Talhinhas and Baroncelli 2021). In the phylogenetic tree, the isolate FAVF642 was placed in the same clade of C. truncatum. Pathogenicity of the isolate FAVF642 was verified on 10 guar seedlings (15-day-old) by spraying a conidial suspension (1 × 106 spores/mL) onto leaves until runoff. Five plants noninoculated served as controls. All plants were kept in a moist chamber for 2 days, and subsequently transferred to a shade house where the temperature ranged from 20 to 30°C. The experiment was conducted twice with similar results. All inoculated leaves developed irregular and necrotic lesions 8 days after inoculation, whereas no symptoms were observed on the control leaves. The fungus was consistently re-isolated from the diseased leaves, fulfilling Koch´s postulates. Colletotrichum truncatum has been previously reported to cause guar anthracnose in India (Farr and Rossman 2022). To our knowledge, this is the first report of C. truncatum causing guar anthracnose in Mexico. This disease is an emerging problem in guar fields in Sinaloa, therefore further studies are required to understand its occurrence and impact in Mexico.

Identification of the GPR55 antagonist binding site using a novel set of high-potency GPR55 selective ligands.

GPR55 is a class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Initially deorphanized as a cannabinoid receptor, GPR55 has been shown to be activated by non-cannabinoid ligands such as l-α-lysophosphatidylinositol (LPI). While there is a growing body of evidence of physiological and pathophysiological roles for GPR55, the paucity of specific antagonists has limited its study. In collaboration with the Molecular Libraries Probe Production Centers Network initiative, we identified a series of GPR55 antagonists using a ß-arrestin, high-throughput, high-content screen of ~300000 compounds. This screen yielded novel, GPR55 antagonist chemotypes with IC50 values in the range of 0.16-2.72 µM [Heynen-Genel, S., et al. (2010) Screening for Selective Ligands for GPR55: Antagonists (ML191, ML192, ML193) (Bookshelf ID NBK66153; PMID entry 22091481)]. Importantly, many of the GPR55 antagonists were completely selective, with no agonism or antagonism against GPR35, CB1, or CB2 up to 20 µM. Using a model of the GPR55 inactive state, we studied the binding of an antagonist series that emerged from this screen. These studies suggest that GPR55 antagonists possess a head region that occupies a horizontal binding pocket extending into the extracellular loop region, a central ligand portion that fits vertically in the receptor binding pocket and terminates with a pendant aromatic or heterocyclic ring that juts out. Both the region that extends extracellularly and the pendant ring are features associated with antagonism. Taken together, our results provide a set of design rules for the development of second-generation GPR55 selective antagonists.

Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands.

Marijuana is the most widely abused illegal drug, and its spectrum of effects suggests that several receptors are responsible for the activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, but the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by their signaling. The orphan receptor GPR55 binds a subset of CB1 and CB2 ligands and has been proposed as a cannabinoid receptor. This designation, however, is controversial as a result of recent studies in which lysophosphatidylinositol (LPI) was identified as a GPR55 agonist. Defining a biological role for GPR55 requires GPR55 selective ligands that have been unavailable. From a ß-arrestin, high-throughput, high-content screen of 300000 compounds run in collaboration with the Molecular Libraries Probe Production Centers Network initiative (PubChem AID1965), we identified potent GPR55 selective agonists. By modeling of the GPR55 activated state, we compared the GPR55 binding conformations of three of the novel agonists obtained from the screen, CID1792197, CID1172084, and CID2440433 (PubChem Compound IDs), with that of LPI. Our modeling indicates the molecular shapes and electrostatic potential distributions of these agonists mimic those of LPI; the GPR55 binding site accommodates ligands that have inverted-L or T shapes with long, thin profiles that can fit vertically deep in the receptor binding pocket while their broad head regions occupy a horizontal binding pocket near the GPR55 extracellular loops. Our results will allow the optimization and design of second-generation GPR55 ligands and provide a means for distinguishing GPR55 selective ligands from those interacting with cannabinoid receptors.

Selection of rhizobacteria isolates with bioherbicide potential against Palmer amaranth (Amarathus palmeri S. Wats.).

Crop yield and quality are affected by the presence of weeds such as Palmer amaranth. Chemical control is the most commonly used method to eradicate weeds, due to its quickness and efficacy. However, the inappropriate use of chemical herbicides can lead to resistant weed biotypes, as well as problems related to environmental pollution and human health hazards. One ecological alternative to combat weeds is the use of deleterious rhizobacteria (DRB). We evaluated the potential bioherbicidal effect in 15 DRB isolates from the rhizosphere of Palmer amaranth, both in vitro and in greenhouse tests. Isolates TR10 and TR18 inhibited seed germination in vitro, whereas the TR25 and TR36 isolates showed the potential to inhibit Palmer amaranth plant development in growth room assays without affecting maize and common bean germination and growth. These four isolates were molecularly identified as either Pseudomonas sp. (TR10 and TR36), Enterobacter sp. (TR18), or Bacillus sp. (TR25). In addition, the production of volatiles and diffusible metabolites were identified as possible mechanisms of germination arrestment and plant development inhibition. This study suggests the bioherbicide potential of some indigenous rhizobacteria against Palmer amaranth.

Genome distribution and validation of novel microsatellite markers of Fusarium verticillioides and their transferability to other Fusarium species.

Improved population studies in the fungus Fusarium verticillioides require the development of reliable microsatellite markers. Here we report a set of ten microsatellite loci that can be used for genetic diversity analyses in F. verticillioides, and are equally applicable to other fungi, especially those belonging to the Gibberella fujikuroi clade.