First report of Pectobacterium polaris causing aerial stem rot of potato in Mexico.

In Mexico, potato (Solanum tuberosum L.) is one of the most important vegetable crops for local consumption and industry. More than 1.8 million tons of potatoes are produced annually, of which the state of Sinaloa contributes with 21.5% (SIAP. 2022). In January 2020, potato plants (cv. FL1867) showing aerial stem rot symptoms were observed in a commercial field from the Santa Rosa Valley, in Northern Sinaloa with an incidence of 36%. Putative pectolytic bacteria showing pitting on crystal violet pectate (CVP) plates were restreaked and purified onto Nutritive Agar (NA) medium at 28°C. Four independent isolates were obtained (L25F-83, L25F-105, L25F-115, and L25F-125) from four symptomatic stems with biochemical and morphological characteristics related to Pectobacterium, such as catalase positive, oxidase negative, pectinolytic activity, Gram-negative and non-fluorescent in B-King medium. Bacterial gDNA was used for amplification and sequencing of two housekeeping genes (dnaX and leuS) (Portier et al. 2019). The nucleotide sequence identity between our isolates was 100% with both housekeeping genes (dnaX, OP376536-OP376539 and leuS, OP376540-OP376543). The BLASTn analysis of dnaX gene shared 98.98% and 99.19% identity with two soft-rot-causing bacterial strains NIBIO1006T (CP017481) and NIBIO1392 (CP017482) of Pectobacterium polaris, respectively; and with leuS gene shared 99.56% identity with P. polaris type strain NIBIO1006T. To further validate the identification, two strains, S5 (isolate L25F-105) and S6 (L25F-125), were selected for whole genome sequencing (WGS). The ANI values for closely related species were calculated using the Orthologous Average Nucleotide Identity (Ortho-ANI) Software Tool (OAT) (Lee et al. 2016). The Type (Strain) Genome Server (TYGS) was used for accurate genome-based taxonomy (https://tygs.dsmz.de) (Meier-Kolthoff et al. 2019). The genomes of P. polaris strains S5 (4811345 pb, GC=52%, AULSZ000000000) and S6 (4809754 pb, GC=52%, JAULTA000000000) revealed 96.86% and 96.07% Ortho-ANI and 73.6% and 66.5% dDDH with P. polaris type strain NIBIO1006T and P. parvum strain CFBP8630, respectively. The MLSA was performed on concatenated complete sequences of dnaX (OR470476, OR470477), leuS (OR470484, OR470485), recA (OR470488, OR470488), acnA (OR470474, OR470475), gapA (OR470478, OR470479), gyrA (OR470480, OR470481), icdA (OR470482, OR470483), proA (OR470486, OR470487), and rpoA genes (OR470490, OR470491). The consensus tree, constructed using the maximum likelihood method (MEGA 7.0), clustered strains S5 and S6 with P. polaris strains NIBIO1006T and NIBIO1392. The four isolates resulted pathogenic in tuber slices and potato seedlings (cv. Fianna) 24 and 72 h, respectively, after being inoculated with 30 µL bacterial suspension (1 X 108 CFU/ml) and incubated at 28 °C and 85% relative humidity. Bacterial colonies were reisolated from the affected tissue and identified with the same PCR primers as described above. Accordingly, P. polaris isolates S5 and S6, fulfill Koch's postulates for aerial stem rot of potato. To our knowledge, this is the first report of P. polaris causing aerial stem rot of potato in Mexico. This bacterium could be a significant threat to the local potato producers; therefore, an accurate and sensitive method of detection and epidemiological studies are needed to support an effective disease diagnosis and management program.

Cucurbit leaf crumple virus (CuLCrV) associated to watermelon disease in Campeche state, Mexico.

An annual recurrent disease causing yield reduction in cultivated watermelon (Citrullus lanatus) was documented by the growers in different farms of Campeche state, Mexico. In April 2019 and March 2020 open field grown watermelon plants showed symptoms such as leaf curling, crumpling, and leaf basal or apical necrosis (Figure S1), with an incidence ranging from 30 up to 80%. These plants also presented high populations of whitefly, especially in the most affected fields. In order to identify the causal agent of the disease, a total of 22 symptomatic watermelon plants were collected in four locations from Campeche state. Total nucleic acids (DNA and RNA) were extracted from these leaf samples. Initially, RT-PCR analysis was performed with specific primers (Table S1) for cucurbit-infecting Crinivirus transmitted by whitefly but the expected size PCR product for those viruses was not amplified in any of these samples. To investigate the presence of cucurbit-infecting begomoviruses, PCR was performed by using specific primers for those begomoviruses reported in Mexico and north/central America including Squash leaf curl virus (SLCV), Watermelon chlorotic stunt virus (WmCSV), Melon chlorotic leaf curl virus (MCLCuV), and Cucurbit leaf crumple virus (CuLCrV) (Table S1). Only the expected amplicon size of ~1089 bp for CuLCrV was amplified from DNA extracts from all 22 watermelon samples, suggesting a single cucurbit-associated virus. The putative complete genome of the CuLCrV Campeche isolate was amplified by circular DNA enrichment using a Rolling Circle Amplification (RCA) procedure from two representative samples, followed by enzymatic digestion using BamHI, EcoRI, KpnI, and SacI enzymes (Inoue-Nagata et al., 2004). Expected linearized full-length viral components (~2.7 kb) were obtained with EcoRI and SacI, and both products, from one selected sample, were cloned in to pGreen0029 vector and were fully sequenced. Sequence analysis of the EcoRI clone, designated as LV2019Camp_A (deposited in GenBank accession no. MW273384) revealed the highest identity of 97.52% to CuLCrV DNA-A isolate Baja California Sur isolate (GeneBank accession no. MN625831.1), whereas the KpnI clone, designated as LV2019Camp_B (deposited in GenBank accession no. MW273385), shared 94.87% identity with DNA B of CuLCrV isolate Arizona (GeneBank accession no. AF327559.1). Subsequently, CuLCrV isolate Campeche-derived agroinfectious clone, was obtained by constructing a partial dimeric tandem repeat of both DNA-A and DNA-B components (Bang et al., 2014). Twelve watermelon plants were agroinfiltrated with the infectious clone at the fourth true leaf stage, resulting in symptomatic plants (11/12) exhibiting leaf yellowing, curling, and crumpling 15 days after agroinfiltrated (Figure S1), and CuLCrV infection was confirmed by PCR specific detection using DNA extract from non-inoculated leaves. Previously CuLCrV has been detected in the USA (Arizona, Texas, California, Florida, South Carolina, and Georgia), and north Mexico (Coahuila) infecting cucurbits including squash, cucumber, cantaloupe, pumpkin, and watermelon (Brown et al., 2000., Keinath et al., 2018), in both single and mixed infection with other whitefly transmitted RNA viruses (CYSDV, genera Crinivirus), and DNA viruses (SLCV, genera Begomovirus) (Kuo et al., 2007). To our knowledge, this is the first report of CuLCrV infecting a cucurbit crop in the Campeche state from the Yucatán peninsula, in Mexico.

Two Populations of Viral Minichromosomes Are Present in a Geminivirus-Infected Plant Showing Symptom Remission (Recovery).

UNLABELLED: Geminiviruses are important plant pathogens characterized by circular, single-stranded DNA (ssDNA) genomes. However, in the nuclei of infected cells, viral double-stranded DNA (dsDNA) associates with host histones to form a minichromosome. In phloem-limited geminiviruses, the characterization of viral minichromosomes is hindered by the low concentration of recovered complexes due to the small number of infected cells. Nevertheless, geminiviruses are both inducers and targets of the host posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) machinery. We have previously characterized a "recovery" phenomenon observed in pepper plants infected with pepper golden mosaic virus (PepGMV) that is associated with a reduction of viral DNA and RNA levels, the presence of virus-related siRNAs, and an increase in the levels of viral DNA methylation. Initial micrococcal nuclease-based assays pinpointed the presence of different viral chromatin complexes in symptomatic and recovered tissues. Using the pepper-PepGMV system, we developed a methodology to obtain a viral minichromosome-enriched fraction that does not disturb the basic chromatin structural integrity, as evaluated by the detection of core histones. Using this procedure, we have further characterized two populations of viral minichromosomes in PepGMV-infected plants. After further purification using sucrose gradient sedimentation, we also observed that minichromosomes isolated from symptomatic tissue showed a relaxed conformation (based on their sedimentation rate), are associated with a chromatin activation marker (H3K4me3), and present a low level of DNA methylation. The minichromosome population obtained from recovered tissue, on the other hand, sedimented as a compact structure, is associated with a chromatin-repressive marker (H3K9me2), and presents a high level of DNA methylation. IMPORTANCE: Viral minichromosomes have been reported in several animal and plant models. However, in the case of geminiviruses, there has been some recent discussion about the importance of this structure and the significance of the epigenetic modifications that it can undergo during the infective cycle. Major problems in this type of studies are the low concentration of these complexes in an infected plant and the asynchronicity of infected cells along the process; therefore, the complexes isolated in a given moment usually represent a mixture of cells at different infection stages. The recovery process observed in PepGMV-infected plants and the isolation procedure described here provide two distinct populations of minichromosomes that will allow a more precise characterization of the modifications of viral DNA and its host proteins associated along the infective cycle. This structure could be also an interesting model to study several processes involving plant chromatin.

A novel tomato spotted wilt virus isolate encoding a noncanonical NSm C118F substitution associated with Sw-5 tomato gene resistance breaking.

The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw-5 resistance gene. Although Sw-5 effectiveness has been shown for most TSWV isolates, the emergence of resistance-breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV-like symptoms were observed in tomato crop cultivars (+Sw-5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118-120 motif and three-dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y-related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full-length genome (TSWV-MX) revealed its reassortment-related evolution and confirmed that putative RB-related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw-5) confirmed the RB nature of TSWV-MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV-RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes.

Gene Expression Profile of Mexican Lime (Citrus aurantifolia) Trees in Response to Huanglongbing Disease caused by Candidatus Liberibacter asiaticus.

Nowadays, Huanglongbing (HLB) disease, associated with Candidatus Liberibacter asiaticus (CLas), seriously affects citriculture worldwide, and no cure is currently available. Transcriptomic analysis of host-pathogen interaction is the first step to understand the molecular landscape of a disease. Previous works have reported the transcriptome profiling in response to HLB in different susceptible citrus species; however, similar studies in tolerant citrus species, including Mexican lime, are limited. In this work, we have obtained an RNA-seq-based differential expression profile of Mexican lime plants challenged against CLas infection, at both asymptomatic and symptomatic stages. Typical HLB-responsive differentially expressed genes (DEGs) are involved in photosynthesis, secondary metabolism, and phytohormone homeostasis. Enrichment of DEGs associated with biotic response showed that genes related to cell wall, secondary metabolism, transcription factors, signaling, and redox reactions could play a role in the tolerance of Mexican lime against CLas infection. Interestingly, despite some concordance observed between transcriptional responses of different tolerant citrus species, a subset of DEGs appeared to be species-specific. Our data highlights the importance of studying the host response during HLB disease using as model tolerant citrus species, in order to design new and opportune diagnostic and management methods.

High-Throughput Sequencing Reveals Differential Begomovirus Species Diversity in Non-Cultivated Plants in Northern-Pacific Mexico.

Plant DNA viruses of the genus Begomovirus have been documented as the most genetically diverse in the family Geminiviridae and present a serious threat for global horticultural production, especially considering climate change. It is important to characterize naturally existing begomoviruses, since viral genetic diversity in non-cultivated plants could lead to future disease epidemics in crops. In this study, high-throughput sequencing (HTS) was employed to determine viral diversity of samples collected in a survey performed during 2012-2016 in seven states of Northern-Pacific Mexico, areas of diverse climatic conditions where different vegetable crops are subject to intensive farming. In total, 132 plant species, belonging to 34 families, were identified and sampled in the natural ecosystems surrounding cultivated areas (agro-ecological interface). HTS analysis and subsequent de novo assembly revealed a number of geminivirus-related DNA signatures with 80 to 100% DNA similarity with begomoviral sequences present in the genome databank. The analysis revealed DNA signatures corresponding to 52 crop-infecting and 35 non-cultivated-infecting geminiviruses that, interestingly, were present in different plant species. Such an analysis deepens our knowledge of geminiviral diversity and could help detecting emerging viruses affecting crops in different agro-climatic regions.