First report of a new Carlavirus provisionally named rose virus B and apple mosaic virus in mixed infections of Rosa sp. in Mexico.

Rose (Rosa sp.) is an important ornamental plant in the cut flower industry around the world. This species is prone to hosting several viruses since it is propagated vegetatively, mainly by grafting (Mollov et al., 2013). In 2021, rose plants of unidentified variety with mosaic, vein yellowing, chlorotic line patterns, and interveinal chlorosis were observed in a rose plantation established in open field in Temixco, Morelos (Supplementary Figure 1). To determine the cause of symptoms was due to viral infection, nucleic acids were extracted from leaves by in-house CTAB procedure and DNase treated. A pooled RNA sample extracted from 4 symptomatic plants was sent to BGI Genomics (China) for high-throughput sequencing (HTS). A stranded mRNA library was prepared and sequenced on the DNBSEQ platform (BGI). A total number of 13,646,715 paired 150-bp clean reads were generated. The reads were assembled de novo into 79,309 contigs ranging from 78 to 15,817 nucleotides (nt) using SPAdes (Prjibelskiet et al., 2020). The contigs were subjected to BLASTx and BLASTn for annotation. A contig with a length of 8,842 nt (208x average coverage per nt) showed 90.6% identity to rose virus B (RVB) (MT473961), and was deposited in GenBank under accession number ON165234. Additionally, three contigs (ON165235-ON165237) corresponding to RNA1 (3,443 nt; 154x coverage), RNA2 (2,938 nt; 231x coverage), and RNA3 (1,897 nt; 232x coverage) of apple mosaic virus (ApMV) were identified. These contigs showed up to 98.4%, 89.7%, and 98.6% identity, respectively, to each corresponding RNA sequences of ApMV. No other viral sequence was identified from the constructed contigs. Subsequently, the presence of RVB was confirmed by RT-PCR performed with an aliquot of the pooled RNAspan style="font-family:'Times New Roman'; font-size:11pt"> with specific primers targeting the replicase and CP (Diaz-Lara et al., 2021). For ApMV, a new set of primers were designed: ApMV_RNA1F (5'-AAATCTCCCGAAAGGGCCTG-3')/ApMV_RNA1R (5'-TCACTCGTCGCATGGATGGATAGC-3'), ApMV_RNA2F (5'-TTGGTACGAGTCGTGGTTGGTTGG-3')/ApMV_RNA2R (5'-GGAAAACTGACCGCAAACCC-3'), and ApMV_RNA3F (5'-GGAGGTTAGAGGCCCGAATG-3')/ApMV_RNA3R (5'-CGCACAGGTGGTAACTCACT-3') which amplify segments of 444 bp, 546 bp, and 434 bp, respectively. The amplicons obtained for both viruses were subjected to Sanger sequencing, confirming the identity of RVB and ApMV. The sequences from the RVB replicase (ON165241) and CP (ON165240) showed 93.9% and 97.0% nt identity with an RVB isolate reported in the USA (MT473961). On the other hand, sequences from RNA1 (ON165238), RNA2, (OP413436), and RNA3 (ON165239) of ApMV had 99.2%, 89.2%, and 99% nt identity, respectively. Finally, the four symptomatic plants were individually tested by RT-PCR to identify RVB and ApMV. Interestingly, both viruses were detected in all the plants analyzed. ApMV (genus Ilarvirus) is associated with mosaic and mottling symptoms in rose (Thomas, 1984). It has been accepted that ApMV is present in rose plants in Mexico (Cardenas-Alonso, 1994), with no evidence to confirm it. RVB was identified in rose in USA, and this virus was classified as a new species of the genus Carlavirus (Diaz-Lara et al., 2021). In addition to RVB, rose virus A and rose virus C have also been reported in rose; however, the symptomatology linked to these viruses is unknown (Xing et al. 2021; Diaz-Lara et al., 2020). Recently, RVB and ApMV were reported in rose plants in Taiwan (Chen et al., 2022). To our knowledge, this is the first report of RVB and ApMV in a mixed infection in rose in Mexico.

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.

Senna multiglandulosa, a new natural host for Tobacco etch virus in Mexico.

Plants of Senna multiglandulosa (family Fabaceae), an ornamental shrub, growing adjacent to tomato and chrysanthemum greenhouses located in San Diego, Texcoco, Estado de Mexico, had leaves with putative virus symptoms, consisting of annular or irregular chlorotic spots of different sizes (Supplementary Fig. 1a). To investigate the presence of a virus, high-throughput sequencing (HTS) was performed. Total RNA was extracted from symptomatic leaves of S. multiglandulosa plants using the SV Total RNA Isolation System Kit (Promega, USA). A portion of the RNA was sent to BGI Genomics (China) for cDNA library construction and sequencing on the DNBSEQ platform (BGI Genomics). HTS yielded 14,673,469 clean paired reads (150x2), which were assembled de novo into 91,879 contigs using SPAdes v3.15 software (Prjibelski et al. 2020). The contigs ranged from 78 to 14,534 nucleotides (nts), which were subjected to BLASTx and BLASTn analyses. A single viral contig of 9,501 nts was detected (average coverage: 56,716x per nt) representing the nearly complete genome of tobacco etch virus (TEV). The highest identity was 79.26% at the nt level (92% query coverage) with TEV isolate TEV7DA (GenBank: DQ986288; length: 9,539 nts) from the USA, and 86.67% at the amino acid (aa) level considering the polyprotein, which are higher than the species demarcation threshold (<76% nt and <82% aa) for the genus Potyvirus (Inoue-Nagata et al. 2022). Additionally, the sequence obtained from S. multiglandulosa revealed 79.21-79.37% nt identities with different TEV isolates from Solanaceae plants (Capsicum annuum, MW748496; Solanum lycopersicum, OM471966.1; Nicotiana tabacum, OL311684.1). The new TEV genome was deposited in GenBank under accession number ON110203. The results obtained by HTS were confirmed by RT-PCR with the original isolated RNA using a pair of specific primers designed from the TEV sequence (TEV-NIb-F, 5'- GCGCTTAAATGCAGACTCGG-3' and TEV-NIb-R, 5'-GTGAAAGTTCAGCAGCAAGCGCA-3') that amplify a 550-bp fragment of the RNA-dependent RNA polymerase. The obtained amplicon was sequenced by the Sanger method, and was 100% identical to the sequence generated by HTS. Subsequently, N. tabacum and N. glutinosa plants were mechanically inoculated using TEV-positive S. multiglandulosa leaves as the inoculum source. Twenty days after inoculation, light chlorotic spots and necrotic lesions were observed on N. tabacum and mosaic on N. glutinosa (Supplementary Fig. 1b-c). RT-PCR analysis confirmed the presence of TEV infection in these indicator plants. To determine the incidence of S. multiglandulosa plants showing TEV-infection symptoms, a survey (n=16) was carried out on two farms in Texcoco; the survey showed a 100% incidence of symptoms. Five survey samples were randomly selected, and the presence of TEV was confirmed by RT-PCR. The discovery of Tobacco etch virus (family Potyviridae: genus Potyvirus) in tobacco was reported in Kentucky, USA in 1928 (Valleau and Johnson, 1928), one of the most common and damaging viruses for the chili crop in Mexico (Delgado, 1974). TEV causes heavy yield loss in several Solanaceae plants and infects more than 120 species in 19 families of dicotyledons (Holmes, 1946). S. obtusifolia (originally Cassia obtusifolia) was the first legume reported as a natural host of TEV in Florida, USA (Anderson, 1954). To our knowledge, this is the first report of the natural infection of S. multiglandulosa by TEV in Mexico and the first TEV genome isolated and sequenced from a legume. S. multiglandulosa is widely distributed in 16 states in Mexico, both cultivated and naturalized, however, it is not considered native to the country (Rzedowski and Calderón, 1997). The occurrence of TEV in S. multiglandulosa represents an alternative reservoir of the virus, with an important role in the epidemiology of the disease.

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

The state of Puebla is the main producer of cabbage (Brassica oleracea var. capitata) in Mexico, with an area of approximately 1,858 ha (SIAP 2023). In April 2023, a field sampling was conducted in the San Luis Ajajalpan, Tecali de Herrera (18°55.57'N, 97°55.607'W), Puebla, Mexico. The average temperature was 24°C and the relative humidity was 95% for five consecutive days. Cabbage plants cv. 'American Taki San Juan' close to harvest, with head rot symptoms were found in a commercial area of approximately 3 ha, at an estimated incidence of 35 to 45%. More than 70% of the leaves were symptomatic on severely affected plants. Typical symptoms included chlorosis of older foliage, soft rot with abundant white to gray mycelium, and abundant production of large and irregularly-shaped sclerotia. The fungus was isolated from 30 symptomatic plants. Sclerotia were collected from symptomatic heads, surface sterilized in 3% NaOCl, rinsed twice with sterile distilled water, 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 placed in an incubator at 25°C in the dark. A total of 30 representative isolates were obtained by the hyphal-tip method, one from each diseased plant (15 isolates from sclerotia and 15 from mycelial fragments). After 8 days, colonies had fast-growing, dense, cottony-white aerial mycelium forming irregular sclerotia of 3.75 ± 0.8 mm (mean ± standard deviation, n=100). Each Petri dish produced 14-25 sclerotia (mean = 18, n = 50), after 10 days. The sclerotia were initially white and gradually turned black. The isolates were identified as Sclerotinia sclerotiorum based on morphological characteristics (Saharan and Mehta 2008). Two representative isolates were chosen for molecular identification, and genomic DNA was extracted by a CTAB protocol. The ITS region and the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene were sequenced for two isolates (White et al. 1990; Staats et al. 2005). The ITS and G3PDH sequences of a representative isolate (SsC.1) were deposited in the GenBank (ITS- OR286628; G3PDH- OR333495). BLAST analysis of the partial sequences ITS (509 bp) and G3PDH (915 bp) showed 100% similarity to S. sclerotiorum isolates (GenBank: MT436756.1 and OQ790148). Pathogenicity was confirmed by inoculating 10 detached cabbage heads of 'American Taki San Juan', using the SsC.1 isolate, according to Sanogo et al. (2015). Heads were placed on the rim of a plastic container and inserted in a moisture box with 2 cm of water on its bottom. The box was covered with a plastic sheet to maintain humidity. The control plants were inoculated with a plug of noncolonized PDA. The inoculated cabbages were covered with white to gray mycelia and abundant sclerotia within 10 days, whereas no symptoms were observed on non-inoculated controls. The fungus was re-isolated from the inoculated cabbages as described above, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. White mold caused by S. sclerotiorum on Brussels sprouts was recently reported in Mexico (Ayvar-Serna et al. 2023). In 2015, S. sclerotiorum was reported on cabbage in New Mexico, causing head rot (Sanogo et al. 2015). To our knowledge, this is the first report of S. sclerotiorum causing white mold on cabbage in Mexico. This research is essential for designing 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.