FIRST REPORT OF BASAL ROT CAUSED BY Fusarium equiseti IN ONION CROPS FROM PUEBLA, MEXICO †

Background : Species of the Fusarium genus are considered as devastating phytopathogens of onion crops around the world. Objective : This work aimed to know the causal agent of basal rot in onion crops from Puebla-México recorded in 2019. Methodology : The causal agent was isolated from diseased samples by tissue incubation in Potato Dextrose Agar medium (PDA) and the pathogenicity tests were done with the causal agent to demonstrate its involvement in basal rot. Monosporic cultures of the causal agent were generated for further microscopic characterization and molecular identification by Internal Transcribed Spacers ITS1 and ITS2. Results : According to the pathogenicity tests, the causal agent produced apical constriction and necrosis in the radicle and leaves accompanied by brown spots surrounded by yellowing as those observed in natural conditions. A 533 bp amplicon of the causative agent was obtained by partial amplification of the 5.8S rDNA gene. The sequence of the amplicon was compared with the sequences deposited in the database of the National Center for Biotechnology Information (NCBI) showing 100% homology with Fusarium equiseti . Implications : Our investigation reveals F. equiseti as an emergent causal agent of onion basal rot in crops from the community of “La Soledad” Puebla, México. Conclusion : Herein we report for the first time F. equiseti as a new phytopathogen of onion and further strategies should be considered for its control.


INTRODUCTION
Onion (Allium cepa L.) is considered one of the most consumed vegetables worldwide whereas in Mexico it is considered an iconic condiment for Mexican cuisine (Joaheer et al., 2019). The production of this food in the country earns about 26,029,376 USD per year and the state of Puebla, México is regarded as the fifth producer, yielding 21,371 tons per hectare (Joaheer et al., 2019).
The Fusarium genus is comprised of thousands of species with several clonal lineages and these species are mainly distributed in the soils, or they are associated with plants as endophytes or potential phytopathogens (Michielse and Rep, 2009;Summerell et al., 2010). Basal rot caused by the genus Fusarium spp. is widely distributed around the globe and has become a limitation in onion and garlic producing areas (Kiehr and Delhey, 2015). The main species within the genus Fusarium that harm the onion crop around the world are F. proliferatum, F. solani and F. oxysporum, reducing its yield up to 50% (Haapalainen et al., 2016). These species produce symptoms in the onion plant that include wilting, rotting of the roots and basal blade of the bulb (Sanogo and Zhang, 2015). In Mexico there are few studies related to the presence of some Fusarium species in onion crops. However, these have been associated with devastating loses for local producers (Montes-Belmont et al., 2010). In the same context, Pulido-Herrera et al. (2008) reported serious root rot incidence caused by Fusarium oxysporum, F. subglutinans and Pyrenochaeta terrestris in onion crops at the Trinidad Valley, in Baja California-Mexico. Due to this fact, the present investigation focused on the identification of the causal agent of the basal rot of in onion crops that emerged in the community of "La Soledad" Puebla, México. The identification of the causal agent was obtained through pathogenicity tests and molecular techniques.

MATERIALS AND METHODS
In the summer of 2019, onion crops (var. 'Crystal white') grown in the community of "La Soledad" Puebla, México (18°27'39.3258"N; -98°37 '11.2614"W) experienced a devastating rot. The symptoms were basal rot, bulb rot, poor root development, leaf discoloration, chlorosis and necrosis in the central part of the leaf ( Figure 1a). Approximately, 40% of the crops showed these symptoms.
Samples of diseased tissues (rot discs and bulbs) were collected in an onion plot (var. 'Crystal white') of 3,144.3 m 2 located in "La Soledad" Puebla, México. This geographical area has a warm-wet climate (cw), with an annual rainfall average of 1,500 mm and an altitude of 1,090 masl. The diseased crops showed loss of leaf turgor, weakness and wilting. The samples were kept at 4 °C and transported in plastic bags to the laboratory for immediate analysis. Thirty bulbs were cut into small pieces (~1.5 cm) and the surface was sterilized with 0.1% sodium hypochlorite for 1 min. The pieces were washed three times with sterile distilled water and dried with sterilized filter paper. Pieces of 0.5 cm 2 were placed in Petri dishes containing PDA medium and incubated for 10 days under 8 h natural light (day) and 16 h darkness at 28 °C). The colonies were purified using monosporic cultures which were maintained in 20% glycerol at -84 °C (Morales-Mora et al., 2020).
The characterization was carried out through fungal microcultures that were visualized in a Carl Zeiss®, (Jena, Germany) at 1000x. Anamorphic structures with morphological characteristics associated with the Fusarium genus were observed, measured and compared with dichotomous keys (Barnett and Hunter, 2006;Leslie and Summerell, 2006). Forty-five certified onion plants of the "Crystal white" var. with a germination percentage of 90% were used for pathogenicity tests. Plants of 30 days old (10 cm tall and 5 mm in diameter) were individually placed in a plastic pot (1 L) containing a sterilized mixture of peatmoss and Agrellite (1:1 v/v) (Martínez-Salgado et al. 2021). The plants were kept under greenhouse conditions (70% RH, 28 °C) in two separate areas. The inoculation of the "CFbC" strain (F. equiseti) was done in these plants by spraying a suspension of 1×10 5 conidia/mL until dropping. The micro-and macroconidia were obtained from cultures developed in PDA and were recovered with 10 mL of sterile physiological saline solution in a laminar flow hood to be incubated for 7 days at 28 °C (Morales-Mora et al., 2019). Fifteen seedlings were only sprayed with distilled water and kept under the same conditions. Genomic DNA was extracted from mycelium of a monosporic culture grown for 7 days in PDA by the CTAB method (Rivera-Jiménez et al., 2018). The genetic material was resuspended in 100 μL HPLC water and immediately quantified by spectrophotometry (Nanodrop 2,000 C, Thermo Scientific®) at 260/280 and 230/260 nm. Afterwards, the genetic material was diluted to a final concentration of 20 ng mL -1 and used as a template for PCR reactions. The amplicons were obtained using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) reported by White et al. (1990) to amplify a partial fragment of the ITS region. PCR mixtures (50 μL) consisted of 20 ng/μL template, 20 μM primers, 500 mM KCL2, 100 mM Tris HCl (pH 9), 50 μM MgCl2, 100 μM dNTPs and 2.5 U/μL Taq DNA polymerase (Promega®). The amplification protocol was performed in accordance with Salazar-González et al. (2016). The amplicons were purified using the kit ExoSAP-IT (Affymetrix®, Santa Clara, CA), following the manufacturer's instructions. The "CFbC" strain were sequenced using the kit BigDye Terminator v3.1 (Applied Biosystems®, Carlsbad, CA) in an Applied Biosystems 3130 sequencer (Carlsbad®, CA) (Juárez-Vázquez et al. 2019). Both complementary chains were assembled and edited using the software BioEdit v7.0.5. As a result, a consensus sequence was obtained for the strain CFbC. The phylogenetic analysis of the strain CFbC was performed by the neighbor-joining method using the MEGA X (Kumar et al., 2018) and the results were compared with five records of the nucleotide database of the National Center for Biotechnology Information.

RESULTS AND DISCUSSION
Ten representative fungi were isolated from 50 diseased onion bulbs. These isolates showed typical morphological features of F. equiseti including white mycelium with radial growth ( Figure 1e). Nevertheless, the isolate named "CFbC" was the most abundant in all samples analyzed. After 11 days, the fungus turned the PDA medium to brown-orange color, which was observed at the bottom of the Petri dish (Figure 1f). The microscopic features of F. equiseti CFbC showed septate hyaline hyphae, septate macroconidia (with five septa) (Figure 1c-d), falcate shaped conidia with a curvature of 60-120 µm (n = 80); the curvature was arcuate in its ventral zone and the dorsal arcs show a prominent basal cell with foot shape and a filamentous apical termination. Microconidia were unicellular, no septate, hyaline and ellipsoid that measured 4.6-17.2 × 1.4-4.1 μm (n = 80). The morphological characteristics coincided with that described in previous studies for F. equiseti (Barnett and Hunter, 2006;Leslie and Summerell, 2006;Summerell et al., 2010).
After 20 days post-inoculation, infected plants exhibited brown spots surrounded by yellowing, necrosis of the root, constriction of apical shoot and wilt of young leaves (Figure 1b). Plants treated with distilled water remained asymptomatic. The fungal pathogen was re-isolated from the lesions and after examination, it exhibited the same morphological characteristics as those of the original isolate. These procedures fulfilled the criteria of Koch's postulates an endorsed F. equiseti as a causal agent of onion basal rot.
The corresponding ITS region was deposited in the nucleotide database of the NCBI with the accession code MN612793. After comparing the sequences of ITS region, the analysis revealed that this sequence had 100% homology with those of the accessions KX375792.1, HM999942.1, KM246255.1, KY554857.1, MG734215.1 and MH860607.1 of F. equiseti (Figure 2). To the best of our knowledge, this is the first report of F. equiseti as an emergent phytopathogen of onion var. 'Crystal white' detected in crops from the community of "La Soledad" Puebla, México. Nevertheless, F. equiseti has been reported as a common causal agent of watermelon rot (Li and Ji et al., 2015). Delgado-Ortiz et al. (2016), isolated and identified several phytopathogenic species, such as F. oxysporum, F. proliferatum, F. verticillioides, F. solani and F. acuminatum in onion crops from the states of Zacatecas and Aguascalientes, Mexico. Also, Fusarium oxysporum, F. proliferatum and F. redolens have been reported as phytopathogens in onion crops from Finland (Haapalainen et al., 2016). Dauda et al. (2018) reported F. equiseti as the causal agent of the dieback of onion crops from Nigeria and Bayraktar et al. (2010) confirmed 40% mortality in onion crops from Turkey caused by the same 4 phytopathogen. On the other hand, Ignjatov et al. (2015) reported the strain FIESC-3 of Fusarium sp. as part of the F. incarnatum-equiseti complex and its involvement in onion seed rot in crops from Serbia.  This research is the first evidence on the presence of F. equiseti as a causal agent of basal rot in onion var. 'Crystal white' from cultures developed in Puebla, Mexico.

CONCLUSION
The morphological and molecular data presented in this work, revealed F. equiseti as a new phytopathogen of onion crops from Puebla, México. Further strategies should be considered for its control.