Metagenomic Analysis of Rhizospheric Bacterial Community of Citrus Trees Expressing Phloem-Directed Antimicrobials.

Huanglongbing, also known as citrus greening, is currently the most devastating citrus disease with limited success in prevention and mitigation. A promising strategy for Huanglongbing control is the use of antimicrobials fused to a carrier protein (phloem protein of 16 kDa or PP16) that targets vascular tissues. This study investigated the effects of genetically modified citrus trees expressing Citrus sinensis PP16 (CsPP16) fused to human lysozyme and ß-defensin-2 on the soil microbiome diversity using 16S amplicon analysis. The results indicated that there were no significant alterations in alpha diversity, beta diversity, phylogenetic diversity, differential abundance, or functional prediction between the antimicrobial phloem-overexpressing plants and the control group, suggesting minimal impact on microbial community structure. However, microbiota diversity analysis revealed distinct bacterial assemblages between the rhizosphere soil and root environments. This study helps to understand the ecological implications of crops expressing phloem-targeted antimicrobials for vascular disease management, with minimal impact on soil microbiota.

Agave attenuata and Agave amica, new hosts of Tuberose mild mosaic virus in Mexico.

Agave attenuata is a Mexican wild plant originally from highlands in the central and occidental mountains of Mexico. This species, known as "swan´s neck agave", is used only as an ornamental plant in public and private gardens. No virus had previously been reported from A. attenuata before this study. In a survey conducted in a commercial greenhouse in Cuautla, Morelos, in 2018, several plants were observed with symptoms of green mosaic and streaks, consistent with a putative viral infection. Sap inoculation from symptomatic A. attenuata plants to herbaceous indicator plants (Nicotiana benthamiana and N. tabacum) failed to produce symptoms in the mechanically inoculated plants. ELISA specific test to CMV, TEV, AMV, TMV and Potyvirus Group (Agdia, Inc.), was positive only for the last one (Chen and Chang, 1998). To determine the identity of the potyvirus involved, total nucleic acid extracts from 100 mg of symptomatic leaves (Trizol reagent; Gibco BRL Life Technologies, England) were used as template in RT-PCR with genus-specific potyvirus primers POT1-POT2, which targeted the variable 5´ terminal half of the coat protein gene of potyviruses (Colinet et al. 1998). The expected 900 bp amplicon was consistently detected in 10 symptomatic A. attenuata plants whereas no PCR products were obtained from 15 asymptomatic A. attenuata plants collected from the "Agaves de México" section at the 'Botanic Garden' of the Instituto de Biología de la UNAM, México. The amplicons were sequenced by the Sanger´s method and the obtained nucleotide (nt) sequences (Acc. No KY190217.1; OP964597-598) and their derived amino acid (aa) sequences were 94.68% to 95.80% similar to an isolate of Tuberose mild mosaic virus (TuMMV; Potyvirus; (Acc. No ON116187.1) characterized from Agave amica in India (Raj et al. 2009). Interestingly, A. amica (formerly Poliantes tuberose) is also a wild Mexican plant that is geographically distributed in the central and south regions of Mexico and is currently being commercially cultivated as an ornamental plant. Plants of A. amica (n=10) showing yellow mild streak were collected from commercial greenhouse and tested positive for TuMMV by RT-PCR and Sanger sequencing (No Acc. OP964599-601 levels) described above. The derived TuMMV sequences from A. attenuata and A. amica were 99-100% similar to each other at the nt/aa level. To exclude the involvement of additional viral agents in the disease, high-throughput sequencing analysis was performed separately for each species of Agave on total RNA extracts from a composite sample of symptomatic leaf tissues using Illumina´s Next Seq 500 platform. Analysis of the obtained 13,260,700 reads (each 75 nt) by the Trinity software, with a total number of sequences of 22,793, resulted in the identification of a single viral contig of 9500 nt for A. attenuata (Acc. No OP964595). Similarly, for A. amica, 27,262,248 reads were obtained, with a total number of sequences of 23,269, resulting in the identification of a single viral contig of 8500 nt (ACC. No OP964602). These contigs showed an identity percentage of 96%/88% and 98%/96% for nucleotides and amino acids, respectively, compared to an isolate of TuMMV from India (Acc. OM293939). Mexico is a center of origin for numerous species of genus Agave which have high economic, social, and ecological impact. TuMMV could be a threat to these plants and potentially to other unknown susceptible crops. To our knowledge, this is the first report of TuMMV in A. attenuata and A. amica in Mexico. REFERENCE Chen, C. C., and Chang, C. A. 1998. Characterization of a potyvirus causing mild mosaic on tuberose. Plant Dis. 82:45-49. Colinet, D., Nguyen, M., Kummert, J., Lepoivre, P., and Xia, F. Z. 1998. Differentiation among potyviruses infecting sweet potato based on genus- and virus-specific reverse transcription polymerase chain reaction. Plant Dis. 82:223-229. Raj, S.K., Snehi, S.K., Kumar, S., Ram, T. and Goel, A.K. 2009. First report of Tuberose mild mosaic potyvirus from tuberose (Polianthes tuberosa L.) in India. Australasian Plant Dis. Notes 4, 93-95.

Bioinformatic-based approach for mutagenesis of plant immune Tm-22 receptor to confer resistance against tomato brown rugose fruit virus (ToBRFV).

Nucleotide-binding leucine-rich repeat (NLR) plant immune receptors mediate the recognition and activation of defense signaling pathways in response to intra- and extracellular pathogens. Several NLR such as Tm-2 and Tm-22 have been introgressed into commercial solanaceous varieties to confer protection against different tobamoviruses. Particularly, Tm-22 was used during recent decades to confer resistance against tobacco mosaic virus, tomato mottle mosaic virus and tomato mosaic virus, which recognizes the viral movement protein (MP). However, tomato brown rugose fruit virus(ToBRFV), a novel tobamovirus, can avoid the protection conferred by Tm-22 due to the presence of key substitutions in the MP. The aim of this work was to identify the key amino acid residues involved in the interaction between Tm-22 and ToBRFV MP through bioinformatic analyses, and to identify potential Tm-22 mutations that could generate greater binding affinity. In silico 3D structure prediction, molecular docking, and computational affinity methods were performed. We predicted that R350, H384 and K385 Tm-22 residues are relevant for the interaction with MP, and two mutations (H384W and K385L) were identified as putative sites to increase the affinity of Tm-22 to the MP with the potential elicitation of resistance against ToBRFV.

Development of a droplet digital polymerase chain reaction (ddPCR) assay for the detection of Tomato brown rugose fruit virus (ToBRFV) in tomato and pepper seeds.

In the present study, a droplet digital PCR assay was developed for detection of Tomato brown rugose fruit virus, a new Tobamovirus of tomato and other solanaceous plants, which expands the diagnostic strategies for this pathogen. Candidate reference DNA material was also obtained to be employed as positive control in tomato and pepper samples. Recombinant plasmids encode for ToBRFV coat protein (CP-ToBRFV) gene and Solanum lycopersicum GAPDH fragments, and CP-ToBRFV and Capsicum annuum GAPDH. To our knowledge, this is the first report of ToBRFV detection in tomato and pepper seeds using ddPCR.

Identification and genomic characterization of a novel tobamovirus from prickly pear cactus.

In this work, we describe the complete sequence and genome organization of a novel tobamovirus detected in a prickly pear plant (Opuntia sp.) by high-throughput sequencing, tentatively named "opuntia virus 2". The full genome of opuntia virus 2 is 6,453 nucleotides in length and contains four open reading frames (ORFs) coding for the two subunits of the RNA polymerase, the movement protein, and the coat protein, respectively. Phylogenetic analysis using the complete nucleotide sequence revealed that the virus belongs to the genus Tobamovirus (family Virgaviridae), showing the highest nucleotide sequence identity (49.8%) with cactus mild mottle virus (CMMoV), being indicating that it belongs in the Cactaceae subgroup of tobamoviruses.

Host-specific loss of sequences of an alfalfa mosaic virus isolate during systemic infection.

Infectivity of an alfalfa mosaic virus (AMV) isolate from Leonotis nepetaefolia in different tomato cultivars was analyzed. Symptoms typical of AMV infection were observed in indicator plants, but not in Flora Dade and Rio Grande tomato cultivars; however, mild symptoms were observed in cv. Rutgers. Furthermore, at least 1 kb of the 3´ segment of RNA 2 and the coat protein gene were missing in systemic leaves of inoculated Rio Grande and Flora Dade plants, while in cv. Rutgers infected with this AMV strain all genomic components were detected. Northern blot analysis of plants infected with the aforementioned AMV isolate confirmed the absence of the CP gene, but suggested rearrangements in both RNA 2 and 3. Factors that may affect differential movement or systemic accumulation of genomic components in multipartite viruses in plants are discussed.

Molecular characterization of a new trichovirus from peach in Mexico.

The complete genome sequence of a trichovirus was obtained from peach samples collected from Mexico and found to be 7985 nucleotides long, excluding the poly(A) tail. Phylogenetic analysis using the complete nucleotide sequence revealed that the virus is a member of the genus Trichovirus and is closely related to peach mosaic virus (PcMV) and cherry mottle leaf virus (CMLV). The highest nucleotide sequence identity was 70% to both PcMV and CMLV, indicating that this virus, which we have tentatively named "peach virus M" (PeVM) should be considered a member of a new trichovirus species. We determined, for the first time, the initiation sites of the subgenomic RNAs (sgRNA) of a trichovirus. The sgRNAs for the movement and coat proteins started with the sequence 'GAA', while the smallest one, coding for the nucleotide-binding protein, started with the nucleotides 'GU'. In all cases, the sgRNAs leader ranged between 113 and 121 nt in length.

Pectobacterium carotovorum subsp. brasiliense Causes Soft Rot and Death of Neobuxbaumia tetetzo in Zapotitlan Salinas Valley, Puebla, Mexico.

Neobuxbaumia tetetzo (Coulter) Backeberg (tetecho) is a columnar cactus endemic to Mexico. Tetecho plants, flowers, fruits, and seeds play an important role in the semiarid ecosystem, as they serve as a refuge and food for insects, bats, and birds, and are widely used by ethnic groups since pre-Hispanic times. Tetecho is affected by a soft rot that damages the whole plant and causes its fall and disintegration. Eight bacterial colonies of similar morphology were isolated from plants showing soft rot and inoculated in healthy tetecho plants, reproducing typical symptoms of soft rot 9 days after inoculation. Ten representative isolates were selected for phenotypic and genetic identification using 16s rDNA, IGS 16S-23S rDNA, and rpoS genes and for pathogenicity tests on several members of the cactus family and other plants. Based on the results, these bacterial isolates were identified as Pectobacterium carotovorum subsp. brasiliense. Inoculation of this bacteria caused soft rot in different cacti, fruits, leaves, and roots of other plants. This is the first report of the subspecies brasiliense of P. carotovorum causing soft rot and death in cacti in the world and the first report of this subspecies in Mexico.

First Report of Hypoxylon diatrypeoides Inducing Dieback and Black Trunk Rot on Mesquite (Prosopis laevigata) in Mexico.

During 2001, branch dieback, black trunk rot, and resinosis were observed on mesquite in the biosphere reserve of Tehuacan, Mexico (18°15'N, 97°25'W) A light brown growth, which included Nodulosporium-like conidiosphores and hyaline conidia that were green in mass and ellipsoid with one end truncate developed on diseased branches. Below the conidiophores and conidia, glomerate to pulvinate stromata formed with conspicuous, black, perithecial mounds with globose perithecia. Ascospores were dark brown, unicellular, ellipsoid, nonequilateral, with narrowly rounded ends, a straight germ slit with a perispore that was dehiscent in 10% KOH, and a conspicuous coil-like, smooth epispore. Sexual reproduction was induced on sterile toothpicks in potato dextrose agar, malt extract agar, or V8 agar (with 10% calcium chloride). The fungus was identified as Hypoxylon diatrypeoides Rehm (1). Samples of mesquite branches with stromata of H. diatrypeoides were deposited in the J. H. Miller Herbarium of the University of Georgia (GAM16048). During the summer of 2002, three pathogenicity tests were performed under greenhouse conditions using three healthy young mesquite plants (25 cm high) per treatment per test. The treatments were: (i) inoculation of branches by wounding with a colonized toothpick from V8 agar, covered with mycelium and perithecia; (ii) spraying ascospores on branches previously wounded with a sterile toothpick; (iii) spraying ascospores on unwounded plants; (iv) plants wounded with sterile toothpicks; and (v) unwounded and uninoculated plants. Fifteen days after inoculation, branch dieback and black trunk rot symptoms were induced in 100% of mesquite plants inoculated with toothpicks and in 50% of wounded plants inoculated with ascospores. No symptoms were seen in the unwounded plants and control treatments. H. diatrypeoides was reisolated from the symptomatic branches. Previously, the fungus had been reported only from the Southern Hemisphere (Brazil and New Zealand), but to our knowledge, this is the first report from Mexico and the Northern Hemisphere. This is also the first evidence of its role as a plant pathogen. Reference: (1) Y. M. Ju and J. D. Rogers. A revision of the genus Hypoxylon. Mycologia Memoir No 20. The American Phytopathological Society, St. Paul, MN, 1996.