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.

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.

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.

Antioxidant and antimutagenic activities of Randia echinocarpa fruit.

We report for the first time the antioxidant and antimutagenic activities of fractions from Randia echinocarpa fruit, which is a Rubiaceae plant native to Sinaloa, Mexico. This fruit has been traditionally used in the prevention or treatment of cancer, among other diseases. The pulp of the fruit was sequentially extracted with solvents of different polarity (i.e. hexane, chloroform, methanol and water). A high extraction yield was obtained with methanol (72.17% d.w.). The aqueous extract showed the highest content of phenolics (2.27 mg/g as ferulic acid equivalents) and the highest antioxidant activity based on the beta-carotene bleaching method (486.15). The commercial antioxidant BHT was used as control (835.05). Antimutagenic activity of the aqueous extract (0-500 microg/tube) was evaluated using the Salmonella microsuspension assay (YG1024 strain) and 1-NP as the mutagen (50 and 100 ng/tube). The aqueous extract was neither toxic nor mutagenic and the percentage of inhibition on 1-NP mutagenicity was 32 and 53% at doses of 50 and 100 ng/tube, respectively. The results of the double incubation assay suggest that the extract inhibited the mutagenicity of 1-NP by a combination of desmutagenic and bioantimutagenic effects.