Ocurrence of Colletotrichum siamense Causing Leaf Spots on Soursop (Annona muricata) in Mexico.

Soursop (Annona muricata L: Annonaceae) is a small tropical fruit tree native to South America (Pinto, 2005). The flesh of its fruits is widely used as a main ingredient of pastries, even young fruits are used as a vegetable. In June 2022, leaf spots symptoms were observed on fifty soursop plants in a commercial nursery located in Juan José Ríos (25°45'20"N 108°50'21"W), Ahome, Sinaloa State. The incidence of the disease was 75%, while the severity was 12%. Symptoms were round, small black necrotic spots, that grew up to 6 mm in diameter with brown or gray color at the center. Fungal isolation was done on potato dextrose agar (PDA) and Colletotrichum-like colonies were obtained. Five isolates were recovered and purified by single spore culture and only a single morphotype was observed. One random isolate was selected for pathogenicity tests, morphological and molecular characterization. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Biotic Products Development Center at the National Polytechnic Institute under accession no. IPN 13.0102. Colonies in PDA at 25°C grow at a rate of 9.0-14.0 mm/d. After 14 days, the colony was olive to gray with orange conidial masses, and conidia (n =100) were hyaline, aseptate, cylindrical, and straight with rounded ends, measuring 11.5 to 18.5 and 3.5 to 5.5 µm. Appressoria were melanized and circular or oval in shape, measuring 6.0 to 4.0 µm (n=20). According to the morphological characteristics observed, the isolate was placed tentatively within the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular confirmation, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), partial sequences of actin (ACT) (Weir et al. 2012) and span style="font-family:'Times New Roman'">glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced. Sequences were deposited in GenBank under the accession numbers: ITS, OQ606966; ACT, OQ617292 and GAPDH, OQ617293. A phylogenetic tree including published sequences of the C. gloeosporiodes species complex was constructed according to Talhinhas and Baroncelli (2021) and the isolate IPN 13.0102 was grouped in a clade with the ex-type culture of C. siamense (ICMP18578) and C. pandanicola. However, C. pandanicola was recorded only as an epiphytic fungus occurring on leaves of Pandanus sp. (Pandanaceae) (Tibpromma et al. 2018) and there are no additional reports of this fungus as a plant pathogen on Pandanus or any other plant. Therefore, the isolate IPN 13.0102 corresponds to C. siamense. Pathogenicity was demonstrated by spraying a conidial suspension (1 × 105 conidia/ml) onto four healthy soursop plants, while two control plants were sprayed using sterile distilled water. All plants were kept in a wet chamber for 48 h at 28  2°C and 85% RH. The characteristic symptoms of the disease were observed 14 days after inoculation, while control plants remained healthy. The pathogenicity test was repeated twice obtaining the same results. The morphology of the recovered fungus was consistently identical to that originally isolated from diseased leaves, fulfilling Koch's postulates. Colletotrichum siamense has been previously reported on Anona spp. in Brazil (Costa et al. 2019). To our knowledge, this is the first report of Colletotrichum siamense causing leaf spots on Annona muricata in Mexico. Further studies for monitoring and control strategies of leaf spots on soursop are required.

Occurrence of Colletotrichum truncatum Causing Foliar Spot on Sesame (Sesamum indicum) in Mexico.

Sesame (Sesamum indicum L.) is an oilseed crop that present agronomic advantages and nutritional contributions in regions where water and soil fertility are limiting. In September 2020 and October 2022, anthracnose symptoms were observed on sesame fields in Mocorito (25°29'04"N;107°55'03"W) and Guasave (25°45'40"N;108°48'44"W), Sinaloa, Mexico. The disease incidence was estimated at up to 35 % (10 has) in five fields. Twenty samples were collected with symptoms on the leaves. On leaves, lesions were irregular and necrotic. Colletotrichum-like colonies were consistently isolated on PDA medium and five monoconidial isolates were obtained. One isolate was selected as a representative for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi of the Biotic Product Development Center at the National Polytechnic Institute under the accession number IPN 13.0101. On PDA, colonies were flat with an entire margin, initially white, then dark gray with black acervuli and setae. The growth rate was 9.3 mm/day. Conidia (n=100) on PDA were hyaloamerosporae, 17.5- 22.7 × 3.6-4.5 µm, smooth-walled, falcated and pointed at both ends, with granular content. Acervuli showed setae acicular (2-3 septate setae) tapered to the apex. The mycelial appressoria were brown, obclavate and irregular. Morphological features matched those of the Colletotrichum truncatum species complex (Damm et al. 2009). For molecular 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 sequences were deposited in GenBank under accession nos. OQ214919 (ITS), OQ230773 (ACT), and OQ230774 (GAPDH). BLASTn searches in GenBank showed 100%, 100%, and 100% identity to MN842788 (ITS), MG198003 (ACT), and MF682518 (GAPDH) of C. truncatum, respectively. A phylogenetic tree based on the Maximum Likelihood method and Bayesian Inference including published ITS, ACT, and GAPDH sequence data for C. truncatum species complex was generated (Talhinhas and Baroncelli 2021). In the phylogenetic tree, the isolate IPN 13.0101 was placed in the same clade of C. truncatum. Pathogenicity of the isolate IPN 13.0101 was verified on 15 sesame seedlings leaves (Dormilon variety) (15-day-old) disinfected with sodium hypochlorite and sterile water. Each leave was inoculated with 200 µL of a conidial suspension (1 × 106 spores/mL). Five plants non inoculated served as controls. All plants were kept in a moist chamber for two days, and subsequently transferred to a shade house where the temperature ranged from 25 to 30°C. All inoculated leaves developed irregular and necrotic lesions ten 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. The experiment was conducted twice with similar results. Colletotrichum spp. has been previously reported (Farr and Rossman, 2023) to cause sesame anthracnose in Mexico (Alvarez, 1976), Thailand (Giatgong, 1980) and Cuba (Arnold, 1986), but this is the first report of C. truncatum causing sesame anthracnose in Mexico. This disease is a recurrent problem in sesame fields in Sinaloa, therefore further studies are required to understand its impact.

First Report of Collar Rot Caused by Sclerotinia sclerotiorum on Sesame (Sesamum indicum) in Mexico.

Sesame (Sesamum indicum L.: Pedaliaceae) is the second most cultivated oilseed in Mexico with 80,000 ha per year. The seeds of this crop are used as a condiment, for the extraction of oil, and its medicinal properties. In October 2020, collar rot symptoms were observed in six sesame fields (SOPC-9539 TD variety) located in the Carrizo Valley (26°15'33.1"N; 109°01'37.9"W), El Fuerte, Sinaloa, México. Initially, small brown spots in the basal stem of the infected plants were observed. At advanced stages of the disease, the circumference of stem was necrotic with the presence of white mycelium that extends to the roots. Infected plants were showing symptoms of yellowing, wilting, and finally death. Disease incidence was estimated at 15%, counting the total of diseased plants in five counts done in arbitrary quadrants within the sesame fields. For fungal isolation, stem sections from the symptomatic basal stem were surface disinfected with 1.5% sodium hypochlorite for 2 min, then triple rinsed with sterile distilled water. The tissue sections were dried on sterile blotting paper and plated in Petri dishes with potato dextrose agar (PDA) culture medium. The plates were incubated at 28ºC in darkness for 48 h. Sclerotinia-like colonies were consistently isolated and four isolates from different locations were purified by the hyphal-tip method. Fungal colonies were formed of compact white mycelium, with the formation of sclerotia on the margin of the plate 6 days after inoculating PDA cultures. Sclerotia averaged 3.1 mm in diameter and 0.024 g. One 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. FAVF654. To confirm identification, genomic DNA was extracted from one isolate, and the internal transcribed spacer (ITS) region was amplified by PCR and sequenced directly using the primer pair ITS5/ITS4 (White et al. 1990). The resulting consensus sequence was deposited in GenBank under accession no. ON401416. BLASTn alignments in GenBank showed 100% identity of our sequence with the sequence of the type strain of Sclerotinia sclerotiorum ATCC 46762 (accession no. JX648201). Pathogenicity of the fungus was demonstrated by inoculating healthy sesame plants (Dormilón and SOPC-9539 TD ies), germinated in plastic pots with sterile substrate. Plants were inoculated with the FAVF654 isolate by applying 3 sclerotia at the base of each of the 12 plants. Twelve plants were left uninoculated, which served as controls. All the inoculated plants, of both varieties, developed the characteristic symptoms of the disease 7 days after inoculation, while the control plants remained symptomless. The pathogenicity test was performed twice with the same result. The fungus was reisolated from all the inoculated plants, thus fulfilling Koch's postulates. Sclerotinia sclerotiorum has been reported on sesame plants in Bulgaria and Korea (Farr and Rossman, 2022). To our knowledge, this is the first report of Sclerotinia sclerotiorum causing collar rot in sesame plants in Mexico and the Americas. This disease considerably reduces the yield of sesame; therefore it is necessary to develop effective disease-management strategies.

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.