First Report of Gray Mold Caused by Botrytis cinerea on Dragon Fruit (Hylocereus undatus) in Mexico.

The dragon fruit is native of Mexico, and Puebla is the third-largest producing state (SIAP 2023). In June 2023, field sampling was conducted in El Paraíso, Atlixco (18° 49' 5.275" N, 98° 26' 52.353" W), Puebla, Mexico. The mean temperature and relative humidity were 20 °C and 75% for seven consecutive days. Dragon fruits cv. 'Delight' close to harvest with gray mold symptoms were found in a commercial area of 2 ha, with an incidence of 35 to 40% and an estimated severity of 75% on infected fruit. The symptoms included necrosis at the apex, which later spread throughout the fruit, along with a soft, black rot covered in abundant mycelium and sporulation. The fungus was isolated from 40 symptomatic fruits by disinfesting pieces of necrotic tissue with 3% NaClO for one minute, rinsing with sterile distilled water (SDW), plating on Petri dishes with potato dextrose agar, and incubating at 25 °C in the dark. One isolate was obtained from each diseased fruit by the hyphal-tip method. The colonies were initially white with a growth rate of 1.15-1.32 cm per day and turned gray after 10 days; the mycelium was dense and aerial. Spherical and irregular sclerotia were formed, measuring 0.9-1.4 × 0.6-1.1 mm (n = 100). Each Petri dish produced 56-278 sclerotia (n = 40) after 11 days; these were initially white and gradually turned dark brown. Brown to olive conidiophores were straight, septate, and branched, measuring 1075-1520 × 10-21 µm, with elliptical hyaline to light brown conidia of 6.6-11.5 × 5-8.1 µm (n=100). The isolates were tentatively identified as Botrytis cinerea based on morphological characteristics (Ellis 1971). Two representative isolates were chosen for molecular identification and genomic DNA was extracted by the CTAB protocol. The ITS region and the heat shock protein (HSP60), RNA polymerase binding II (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) genes were sequenced (White et al. 1990; Staats et al. 2005). The sequences of a representative isolate (BcPh5) were deposited in GenBank (ITS-OR582337; HSP60-OR636622; RPB2-OR636623; and G3PDH-OR636621). BLAST analysis of the partial sequences of ITS (479 bp), HSP60 (1006 bp), RPB2 (1126 bp), and G3PDH (907 bp) showed 100% similarity to B. cinerea isolates (GenBank: KM840848, MH796663, MK919495, MF480679). Phylogenetic analysis confirmed that BcPh5 clustered with B. cinerea strains. Pathogenicity was confirmed by inoculating the non-wounded surface of 20 detached dragon fruits cv. 'Delight' using the BcPh5 isolate by depositing 20 µl of a 105 conidia/ml suspension with a sterile syringe. The fruits were placed on the rim of a plastic container and inserted in a moisture box with 2 cm of water at the bottom. The box was covered with a plastic sheet to maintain humidity. Control fruits were inoculated with SDW. The inoculated fruits became covered with abundant white to gray mycelium, and soft rot developed within eight days, while no symptoms were observed on the controls. The fungus was re-isolated from the inoculated fruits as described above, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. Gray mold caused by B. cinerea was also recently reported in Mexico on pomegranate (Hernández et al. 2023) and rose apple (Isodoro et al. 2023). As far as we know, this is the first report of B. cinerea causing gray mold on dragon fruit in Mexico. This research is essential for designing integrated management strategies against gray mold on dragon fruits.

First Report of White Mold Caused by Sclerotinia sclerotiorum on Echeveria gigantea in Mexico.

Echeveria gigantea, native of Mexico (Reyes et al. 2011), holds economic importance as it is marketed as a potted plant and cut flower due to its drought-tolerant capabilities and aesthetic appeal. In September 2023, a field sampling was conducted at the Research Center in Horticulture and Native Plants (18°55'56.6" N, 98°24'01.5" W) of UPAEP University. Echeveria gigantea cv. Quilpalli plants with white mold symptoms were found in an area of 0.5 ha, with an incidence of 40% and severity of 50% on severely affected stems. The symptoms included chlorosis of older foliage, necrosis at the base of the stem, and soft rot with abundant white to gray mycelium and abundant production of irregular sclerotia resulting in wilted plants. The fungus was isolated from 30 symptomatic plants. Sclerotia were collected, sterilized in 3% NaOCl, rinsed with sterile distilled water (SDW), and plated on Potato Dextrose Agar (PDA) with sterile forceps. Subsequently, a dissecting needle was used to place fragments of mycelium directly on PDA. Plates were incubated at 23 °C in darkness. A total of 30 isolates were obtained using the hyphal-tip method, one from each diseased plant (15 isolates from sclerotia and 15 from mycelium). After 6 days, colonies had fast-growing, dense, cottony-white aerial mycelium forming irregular sclerotia of 3.67 ± 1.13 mm (n=100). Each Petri dish produced 32.47 ± 7.5 sclerotia (n=30), after 12 days. The sclerotia were initially white and gradually turned black. The isolates were tentatively identified as Sclerotinia sclerotiorum based on morphological characteristics (Saharan and Mehta 2008). Two isolates were selected for molecular identification. Genomic DNA was extracted using the CTAB protocol. The ITS region and the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene were sequenced for two randomly selected isolates (White et al. 1990; Staats et al. 2005). The ITS and G3PDH sequences of the SsEg9 isolate were deposited in GenBank (ITS-OR816006; G3PDH-OR879212). BLAST analysis of the partial ITS (510 bp) and G3PDH (915 bp) sequences showed 100% and 99.78% similarity to S. sclerotiorum isolates (GenBank: MT101751 and MW082601). Pathogenicity was confirmed by inoculating 30 120-day-old E. gigantea cv. Quilpalli plants grown in pots with sterile soil. Ten sclerotia were deposited at the base of the stem, 10 mm below the soil surface. As control treatment, SDW was applied to 10 plants. The plants were placed in a greenhouse at 23 °C and 90% relative humidity. After 16 days, all inoculated plants displayed symptoms similar to those observed in the field. Control plants did not display any symptoms. The fungus was reisolated from the inoculated stems, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. Recently S. sclerotiorum has been reported causing white mold on cabbage in the state of Puebla, Mexico (Terrones-Salgado et al. 2023). To the best of our knowledge, this is the first report of S. sclerotiorum causing white mold on E. gigantea in Mexico. Information about diseases affecting this plant is very limited, so this research is crucial for designing integrated management strategies and preventing spread to other production areas.

First report of Athelia rolfsii (= Sclerotium rolfsii) causing southern blight on Pachyrhizus erosus in Mexico.

Pachyrhizus erosus, commonly named jicama, is native to Mexico and is cultivated for its tuberous roots which are edible. In November 2021, field sampling was carried out in municipality of Huaquechula (18.748640N, 98.550817W, 1,580 m above sea level), state of Puebla, México. The disease had an incidence between 20 and 30% in approximately 10 ha. Infected plants showed wilting, yellowing foliage, rotting with white mycelium, abundant sclerotia were observed in the roots and tuber. Tuber splits transversely over time. Twenty plants with symptoms of disease were carried out to isolate the fungus. The sclerotia found in the tubers were disinfected with 3% NaOCl, rinsed twice with sterile distilled water, and excess moisture was removed and, transferred on Potato Dextrose Agar (PDA) culture medium and incubated at 28°C. Mycelial fragments from symptomatic tubers, were plated directly to PDA. Twenty representative isolates were obtained by hyphal-tip method, one for each diseased plant sampled (10 isolates from sclerotia and the other 10 from fragments of mycelium). After 10 days, colonies showed fast-growing, dense, cottony-white aerial mycelium, forming globoid to irregular sclerotia, measuring 1.0-1.7 mm in diameter (mean = 1.42 mm; n=100). The number of sclerotia produced per Petri dish ranged from 54 to 542 (mean = 274, n = 50). These sclerotia were initially white and gradually turned brown. Microscopic examination showed septate hyphae with some cells having clamp connections. Based on morphological characteristics, the fungal isolates were identified as Athelia rolfsii (Curzi) CC Tu & Kimbr (Syn: Sclerotium rolfsii Sacc) (Mordue 1974). For molecular identification, a representative isolate (Sr.1), the ITS region was amplified (650 bp) using primers ITS1/ITS4 (White et al. 1990). The obtained sequence (GenBank: ON206899) was subjected to BLAST analysis, where it had 100% identity with A. rolfsii isolates (GenBank: MG836252 and MH517363). Phylogenetic analysis with the neighbor-joining method in MEGAX, grouped the Sr.1 isolate into a common clade with different A. rolfsii isolates. Pathogenicity was confirmed by inoculating 20 tubers detached from healthy P. erosus variety "Criolla de Morelos", into which a portion of mycelium from the Sr.1 isolate was inserted with a sterile wooden stick at one point per tuber. In five tubers, only a sterile wooden stick was inserted as negative controls. The tubers were placed under laboratory conditions with relative humidity close to 100% and a temperature of 28°C. Symptoms like those observed in the field were observed after five days. Control tubers showed no symptoms. Additional pathogenicity tests were performed on 50 plants of 100-day-old P. erosus of the variety "Criolla de Morelos", grown in pots with sterile soil. Ten sclerotia of 10 days old were deposited at the base of the stem, 10 mm below the soil surface; as control treatment only, sterile distilled water was deposited on 20 plants. The plants were placed in a greenhouse (Center for Technological Innovation in Protected Agriculture of the Popular Autonomous University of the State of Puebla), at 28 ± 1°C and 90% of temperature and relative humidity, respectively. After 15 days, all inoculated plants showed symptoms similar to those observed in the field. Control plants showed no symptoms. A. rolfsii was re-isolated from inoculated tubers and stem, fulfilling Koch's postulates. Previously, A. rolfsii was reported in Mexico, causing southern blight on sesame (Hernández-Morales et al. 2018). To our knowledge, this is the first report of Athelia rolfsii causing southern blight on P. erosus in Mexico (Farr and Rossman 2022). This research is important to design management strategies and prevent its spread to other P. erosus-producing areas.

First report of Lasiodiplodia theobromae causing leaf rot on Agave angustifolia in Mexico.

The agave crop (Agave angustifolia), is of economic importance for Mexico, for the agave is made mainly an alcoholic beverage called locally mezcal. In the state of Guerrero, in the municipality of Huitzuco de los Figueroa (18.2510026N, 99.2320182W, 1196 m above sea level), a severe disease affecting agave leaves was detected. The field symptoms consisted of pale to brown dark descending lesions, covering >50% of the leaf surface, in which the presence of pycnidia was observed. In an estimated area of 0.5 ha, the estimated incidence was 67% (n=100 plants). Symptomatic fragments from leaves (approximately 0.5 cm) were taken, superficially disinfected with 1% NaClO, and rinsed twice with sterile distilled water. Then they were transferred to potato dextrose agar (PDA) medium, and incubated at 28 °C. After five days, twelve representative isolates were selected and purified by the hyphal tip technique. In the PDA medium, the colonies were initially light gray, later they became dark, and after 22 days of incubation, the development of numerous dark pycnidia was observed on the surface of the medium. Initially, immature hyaline conidia, unicellular, oval, and double-walled were observed. The mature conidia were dark brown, oval, with one septum and longitudinal striation, and measured 17.5 to 27 [average 25.3 µm; n=50] × 10.5 to 15.7 [average 13.9 µm; n=50]. Based on the morphological characteristics, the fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Alves et al. 2008). Isolates LAS3 and LAS4 were used for molecular identification, this was done by amplifying the regio internal transcribed spacer (ITS) of rDNA with primers ITS1 and ITS4 (White et al. 1990) and translation elongation factor 1-alpha ( EF-1α) genes using primers EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting sequences were deposited in GenBank (LAS3; ON391564 and LAS4; ON391565 for ITS, and LAS3; ON368190 and LAS4; ON368191 for EF-1α). BLASTn analysis sequences of isolated LAS3 and LAS4 revealed for ITS 98.6% identity with L. theobromae (MK934699.1), and for EF-1α indicated 100% identity (MF422024.1). From concatenated sequences ITS-EF-1α regions, a phylogenetic analysis was carried out in MEGA X software, using the Maximum Likelihood and Kimura 2-parameter model with 1,000 bootstraps replicated; isolates LAS3 and LAS4 were clustered in the clade of the members of L. theobromae strains CAA006 (Alves et al. 2006), and INTA-IMC 1601 (Perez et al. 2018). The pathogenicity tests were carried out on 10 healthy 3 year-old agave plants, in which the mycelium of the LAS4 isolate was inserted at three equidistant points/leaf, using a sterile toothpick. Five healthy agave plants were inoculated only with sterile PDA as control treatment. The inoculated plants were covered with transparent plastic bags and housed in a greenhouse at 28 °C. After seven days, similar symptoms to those observed in the field were observed in all inoculated plants. Control plants did not develop symptoms. The fungus L. theobromae was re-isolated again from the infected leaves, fulfilling Koch's postulates. In China, L. theobromae has been reported as the cause of leaf rot on A. sisalana (Xie et al. 2016). To our knowledge, this is the first report of L. theobromae causing leaf rot on A. angustifolia in Mexico. This research is useful to design management strategies for leaf rot disease for local farmers of A. angustifolia.

First Report of Bud Soft Rot on Agave angustifolia Caused by Pantoea dispersa in Mexico.

The agave (Agave spp.) is an important crop in México, with 120,897 ha grown mainly for alcoholic beverage production (SIAP, 2019). In September 2020, in the municipality of Huitzuco de los Figueroa (18.328692 N; 99.3998 W), Guerrero State, México, a serious disease was observed affecting Agave angustifolia. Disease incidence was 8% of 150 plants sampled over an approximate area of 2.5 ha. Initial symptoms of soft rot of the bud developed and produced an abundant exudate which appeared from the apical part to the base of the plant. In severe infections, the plants showed total maceration of the bud, and consequently death of the plants was observed. Symptomatic plant tissue was superficially disinfected with 1% NaOCl for 30 s, and rinsed in sterile water three times. The disinfected tissues were macerated and with a loop spread in Nutrient Agar. The plates were incubated at 28 ° C for 2 days. Yellowish bacterial colonies were isolated, and eight colonies were selected for characterization. The bacterial strains were gram negative and rod-shaped, negative for fluorescent pigment tests and Kovacs' oxidase. Two isolates designated AGA1 and AGA2 were identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F / 1492R (Lane 1991), and partial fusA, rpoB, and gyrB genes (Delétoile et al. 2009). Sequences were deposited in GenBank, with the accession numbers for 16S rRNA, AGA1 as MW548406 and AGA2 as MW548407; for specific genes fusA (AGA1 = MW558445, AGA2 = MW558446), rpoB (AGA1 = MW558447, AGA2 = MW558448) and gyrB (AGA1 = MW558449, AGA2 = MW558450), and they were compared with the sequences available in GenBank using BLASTn. 16S rRNA gene sequences for AGA1 and AGA2 aligned with Pantoea dispersa (MT921704.1, 99.9% identity). Housekeeping genes also aligned 99 to 100% to P. dispersa (fusA = 100%, CP045216.1; rpoB = 99.8% MH015167.1 and gyrB = 99%, MK928270.1). Phylogenetic analysis of concatenated genes showed that strains AGA1 and AGA2 cluster with P. dispersa. To confirm pathogenicity, eight plants of six-month-old A. angustifolia were inoculated with strain AGA1 using sterile toothpicks dipped in 108 CFU/ml bacterial suspension. The toothpicks were inserted in the middle part of the bud. Four plants were inoculated with sterile water as control. The plants were covered with plastic bags and housed in a greenhouse (average temperature and relative humidity of 25 ° C and 85%, respectively). Pathogenicity tests were repeated two times. After seven days, all inoculated plants developed symptoms similar to those observed in the field. Control plants did not show symptoms. From the plants that showed symptoms, the pathogen was reisolated again and was identified by morphological and molecular characterization, following the method previously described, fulfilling Koch's postulates. In México, Erwinia cacticida and Pantoea ananatis has been previously reported on A. tequilana that as causing soft rot and red leaf ring, respectively (Jimenez-Hidalgo et al. 2004; Fucikovsky and Aranda 2006). To our knowledge, this is the first report of P. dispersa causing bud soft rot on A. angustifolia in México. More studies monitoring and control strategies of bud soft rot on A. angustifolia are required.