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.

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.

Differential Expression of miRNAs Involved in Response to Candidatus Liberibacter asiaticus Infection in Mexican Lime at Early and Late Stages of Huanglongbing Disease.

Huanglongbing (HLB) is one of the most destructive diseases threatening citriculture worldwide. This disease has been associated with α-proteobacteria species, namely Candidatus Liberibacter. Due to the unculturable nature of the causal agent, it has been difficult to mitigate the disease, and nowadays a cure is not available. MicroRNAs (miRNAs) are key regulators of gene expression, playing an essential role in abiotic and biotic stress in plants including antibacterial responses. However, knowledge derived from non-model systems including Candidatus Liberibacter asiaticus (CLas)-citrus pathosystem remains largely unknown. In this study, small RNA profiles from Mexican lime (Citrus aurantifolia) plants infected with CLas at asymptomatic and symptomatic stages were generated by sRNA-Seq, and miRNAs were obtained with ShortStack software. A total of 46 miRNAs, including 29 known miRNAs and 17 novel miRNAs, were identified in Mexican lime. Among them, six miRNAs were deregulated in the asymptomatic stage, highlighting the up regulation of two new miRNAs. Meanwhile, eight miRNAs were differentially expressed in the symptomatic stage of the disease. The target genes of miRNAs were related to protein modification, transcription factors, and enzyme-coding genes. Our results provide new insights into miRNA-mediated regulation in C. aurantifolia in response to CLas infection. This information will be useful to understand molecular mechanisms behind the defense and pathogenesis of HLB.

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.

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.

De novo assembly and functional annotation of Citrus aurantifolia transcriptome from Candidatus Liberibacter asiaticus infected and non-infected trees.

Mexican lime (Citrus aurantifolia) belongs to the Rutaceae family and nowadays is one of the major commercial citrus crops in different countries. In Mexico, Mexican lime production is impaired by Huanglongbing (HLB) disease associated to Candidatus Liberibacter asiaticus (CLas) bacteria. To date, transcriptomic studies of CLas-Citrus interaction, have been performed mainly in sweet citrus models at symptomatic (early) stage where pleiotropic responses could mask important, pathogen-driven host modulation as well as, host antibacterial responses. Additionally, well-assembled reference transcriptomes for acid limes including C. aurantifolia are not available. The development of improved transcriptomic resources for CLas-citrus pathosystem, including both asymptomatic (early) and symptomatic (late) stages, could accelerate the understanding of the disease. Here, we provide the first transcriptomic analysis from healthy and HLB-infected C. aurantifolia leaves at both asymptomatic and symptomatic stages, using a RNA-seq approach in the Illumina NexSeq500 platform. The construction of the assembled transcriptome was conducted using the predesigned workflow Transflow and a total of 41,522 tentative transcripts (TTs) obtained. These C. aurantifolia TTs were functionally annotated using TAIR10 and UniProtKB databases. All raw reads were deposited in the NCBI SRA with accession numbers SRR10353556, SRR10353558, SRR10353560 and SRR10353562. Overall, this dataset adds new transcriptomic valuable tools for future breeding programs, will allow the design of novel diagnostic molecular markers, and will be an essential tool for studying the HLB disease.

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.

Multiple phytoplasmas associated with potato diseases in Mexico.

In recent years, the potato crop in Mexico has been notably affected by diseases recognized as potato purple top (PPT) in foliage and potato hair sprouts (PHS) in germinating tubers. In both cases, these syndromes reduce production by affecting viability of the tubers used as seeds. There is evidence indicating that phytoplasmas are associated with these syndromes. This study presents data on the molecular detection, characterization, and ecology of the pathogens related to PPT and PHS. Restriction fragment length polymorphism (RFLP) and sequence analysis indicated that PPT phytoplasma belongs to the 16SrI group and PHS phytoplasma fits in the 16SrII group. In this paper, we report that the two different phytoplasmas have been found coexisting in the same potato plant, which demonstrates the presence of mixed infection in the field. These phytoplasmas were also detected in weeds surrounding potato fields; therefore they should be considered as alternative hosts or natural reservoirs of PPT and PHS phytoplasmas.