Insect vector and reservoir plant of 'Fragaria × ananassa' phyllody phytoplasma (16SrXIII-F) in central region of Chile.

Strawberry phyllody has emerged as a prevalent disease affecting Chilean strawberry in recent years. The causal pathogen, 'Fragaria × ananassa' phyllody phytoplasma (StrPh), is categorized within the 16S ribosomal group XIII, exclusively found in the Americas. In the context of economically significant crops, hemipteran insect vectors and alternative host plants play a pivotal role in their natural dissemination. This study comprehensively examined the key epidemiological facets of StrPh in the central region of Chile: the insect vector and alternative hosts. Through field surveys, we identified an abundance of an insect species, Cixiosoma sp., in an StrPh-infected strawberry field, and confirmed its role as a vector of this phytoplasma through subsequent transmission assays. Moreover, we found a spontaneous weed species, Galega officinalis, to be infected with StrPh, raising the possibility of it being a potential alternative host plant for this phytoplasma. StrPh was also detected in cold-stored strawberry runners purchased from a nursery that supplies the local strawberry cultivation, suggesting a potential source of this phytoplasma in Chile. Collectively, these findings provide a significant epidemiological source of StrPh dissemination in central Chile.

Old and New Aphid-Borne Viruses in Coriander in Chile: An Epidemiological Approach.

In Chile, edible herbs are mainly grown by small farmers. This type of horticultural crop typically requires intensive management because it is highly susceptible to insects, some of which transmit viruses that severely affect crop yield and quality. In 2019, in coriander plants tested negative for all previously reported viruses, RNA-Seq analysis of one symptomatic plant revealed a plethora of viruses, including one virus known to infect coriander, five viruses never reported in coriander, and a new cytorhabdovirus with a 14,180 nucleotide RNA genome for which the species name Cytorhabdovirus coriandrum was proposed. Since all the detected viruses were aphid-borne, aphids and weeds commonly growing around the coriander field were screened for viruses. The results showed the occurrence of the same seven viruses and the alfalfa mosaic virus, another aphid-borne virus, in aphids and weeds. Together, our findings document the presence of multiple viruses in coriander and the potential role of weeds as virus reservoirs for aphid acquisition.

Development of a Genome-Informed Protocol for Detection of Pseudomonas amygdali pv. morsprunorum Using LAMP and PCR.

One of the causal agents of bacterial canker is Pseudomonas amygdali pv. morsprunorum-Pam (formerly Pseudomonas syringae pv. morsprunorum). Recently detected in Chile, Pam is known to cause lesions in the aerial parts of the plant, followed by more severe symptoms such as cankers and gummosis in the later stages of the disease. This study presents the design of PCR and LAMP detection methods for the specific and sensitive identification of Pseudomonas amygdali pv. morsprunorum (Pam) from cherry trees. Twelve Pseudomonas isolates were collected, sequenced, and later characterized by Multi-locus Sequence Analysis (MLSA) and Average Nucleotide Identity by blast (ANIb). Three of them (11116B2, S1 Pam, and S2 Pam) were identified as Pseudomonas amygdali pv. morsprunorum and were used to find specific genes through RAST server, by comparing their genome with that of other Pseudomonas, including isolates from other Pam strains. The effector gene HopAU1 was selected for the design of primers to be used for both techniques, evaluating sensitivity and specificity, and the ability to detect Pam directly from plant tissues. While the PCR detection limit was 100 pg of purified bacterial DNA per reaction, the LAMP assays were able to detect up to 1 fg of purified DNA per reaction. Similar results were observed using plant tissues, LAMP being more sensitive than PCR, including when using DNA extracted from infected plant tissues. Both detection methods were tested in the presence of 30 other bacterial genera, with LAMP being more sensitive than PCR.

Transcriptome Analysis of Sweet Cherry (Prunus avium L.) Cultivar 'Lapins' upon Infection of Pseudomonas syringae pv. syringae.

Bacterial canker caused by Pseudomonas syringae pv. syringae (Pss) is responsible for substantial loss to the production of sweet cherry in Chile. To date, the molecular mechanisms of the Pss-sweet cherry interaction and the disease-related genes in the plant are poorly understood. In order to gain insight into these aspects, a transcriptomic analysis of the sweet cherry cultivar 'Lapins' for differentially expressed genes (DEGs) in response to Pss inoculation was conducted. Three Pss strains, A1M3, A1M197, and 11116_b1, were inoculated in young twigs, and RNA was extracted from tissue samples at the inoculation site and distal sections. RNA sequencing and transcriptomic expression analysis revealed that the three strains induced different patterns of responses in local and distal tissues. In the local tissues, A1M3 triggered a much more extensive response than the other two strains, enriching DEGs especially involved in photosynthesis. In the distal tissues, the three strains triggered a comparable extent of responses, among which 11116_b1 induced a group of DEGs involved in defense responses. Furthermore, tissues from various inoculations exhibited an enrichment of DEGs related to carbohydrate metabolism, terpene metabolism, and cell wall biogenesis. This study opened doors to future research on the Pss-sweet cherry interaction, immunity responses, and disease control.

First Record of Colletotrichum anthrisci Causing Anthracnose on Avocado Fruits in Chile.

Anthracnose caused by Colletotrichum species is one of the most frequent and damaging fungal diseases affecting avocado fruits (Persea americana Mill.) worldwide. In Chile, the disease incidence has increased over the last decades due to the establishment of commercial groves in more humid areas. Since 2018, unusual symptoms of anthracnose have been observed on Hass avocado fruits, with lesions developing a white to gray sporulation. Morphological features and multi-locus phylogenetic analyses using six DNA barcodes (act, chs-1, gapdh, his3, ITS, and tub2) allowed the identification of the causal agent as Colletotrichum anthrisci, a member of the dematium species complex. Pathogenicity was confirmed by inoculating healthy Hass avocado fruits with representative isolates, reproducing the same symptoms initially observed, and successfully reisolating the same isolates from the margin of the necrotic pulp. Previously, several Colletotrichum species belonging to other species complexes have been associated with avocado anthracnose in other countries. To our knowledge, this is the first record of C. anthrisci and of a species of the dematium species complex causing anthracnose on avocado fruits in Chile and worldwide.

Photorhabdus antumapuensis sp. nov., a novel symbiotic bacterial species associated with Heterorhabditis atacamensis entomopathogenic nematodes.

One motile, Gram-negative, non-spore-forming and rod-shaped symbiotic bacterium, strain UCH-936T, was isolated from Heterorhabditis atacamensis nematodes. Results of biochemical, physiological, molecular and genomic analyses suggest that it represents a new species, which we propose to name Photorhabdus antumapuensis sp. nov. Digital DNA-DNA hybridization shows that strain UCH-936T is more closely related to Photorhabdus kleinii DSM 23513T, but shares solely 50.5 % similarity, which is below the 70% cut-off value that delimits species boundaries in bacteria. Phylogenetic reconstructions using whole-genome sequences show that strain UCH-936T forms a unique clade, suggesting its novel and distinct taxonomic status again. Similarly, comparative genomic analyses shows that the virulence factor flagella-related gene fleR, the type IV pili-related gene pilL and the vibriobactin-related gene vibE are present in the genome of strain UCH-936T but absent in the genomes of its closest relatives. Biochemically and physiologically, UCH-936T differs also from all closely related Photorhabdus species. Therefore, Photorhabdus antumapuensis sp. nov. is proposed as a new species with the type strain UCH-936T (CCCT 21.06T=CCM 9188T=CCOS 1991T).

Revision of the 'Candidatus Phytoplasma' species description guidelines.

The genus 'Candidatus Phytoplasma' was proposed to accommodate cell wall-less bacteria that are molecularly and biochemically incompletely characterized, and colonize plant phloem and insect vector tissues. This provisional classification is highly relevant due to its application in epidemiological and ecological studies, mainly aimed at keeping the severe phytoplasma plant diseases under control worldwide. Given the increasing discovery of molecular diversity within the genus 'Ca. Phytoplasma', the proposed guidelines were revised and clarified to accommodate those 'Ca. Phytoplasma' species strains sharing >98.65 % sequence identity of their full or nearly full 16S rRNA gene sequences, obtained with at least twofold coverage of the sequence, compared with those of the reference strain of such species. Strains sharing <98.65 % sequence identity with the reference strain but >98.65 % with other strain(s) within the same 'Ca. Phytoplasma' species should be considered related strains to that 'Ca. Phytoplasma' species. The guidelines herein, keep the original published reference strains. However, to improve 'Ca. Phytoplasma' species assignment, complementary strains are suggested as an alternative to the reference strains. This will be implemented when only a partial 16S rRNA gene and/or a few other genes have been sequenced, or the strain is no longer available for further molecular characterization. Lists of 'Ca. Phytoplasma' species and alternative reference strains described are reported. For new 'Ca. Phytoplasma' species that will be assigned with identity ≥98.65 % of their 16S rRNA gene sequences, a threshold of 95 % genome-wide average nucleotide identity is suggested. When the whole genome sequences are unavailable, two among conserved housekeeping genes could be used. There are 49 officially published 'Candidatus Phytoplasma' species, including 'Ca. P. cocostanzaniae' and 'Ca. P. palmae' described in this manuscript.

Xenorhabdus lircayensis sp. nov., the symbiotic bacterium associated with the entomopathogenic nematode Steinernema unicornum.

Xenorhabdus is a symbiotic group of bacteria associated with entomopathogenic nematodes of the family Steinernematidae. Although the described Steirnernema species list is extensive, not all their symbiotic bacteria have been identified. One single motile, Gram-negative and non-spore-forming rod-shaped symbiotic bacterium, strain VLST, was isolated from the entomopathogenic nematode Steinernema unicornum. Analyses of the 16S rRNA gene determined that the VLST isolate belongs to the genus Xenorhabdus, and its closest related species is Xenorhabdus szentirmaii DSM 16338T (98.2 %). Deeper analyses using the whole genome for phylogenetic reconstruction indicate that VLST exhibits a unique clade in the genus. Genomic comparisons considering digital DNA-DNA hybridization (dDDH) values confirms this result, showing that the VLST values are distant enough from the 70 % threshold suggested for new species, sharing 30.7, 30.5 and 30.3 % dDDH with Xenorhabdus khoisanae MCB, Xenorhabdus koppenhoeferi DSM 18168T and Xenorhabdus miraniensis DSM 18168T, respectively, as the closest species. Detailed physiological, biochemical and chemotaxonomic tests of the VLST isolate reveal consistent differences from previously described Xenorhabdus species. Phylogenetic, physiological, biochemical and chemotaxonomic approaches show that VLST represents a new species of the genus Xenorhabdus, for which the name Xenorhabdus lircayensis sp. nov. (type strain VLST=CCCT 20.04T=DSM 111583T) is proposed.

Survey for 'Candidatus Liberibacter' and 'Candidatus Phytoplasma' in Citrus in Chile.

The considerable economic losses in citrus associated with 'Candidatus Liberibacter' and 'Candidatus Phytoplasma' presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of 'Ca. Liberibacter' associated with "huanglongbing" disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phytoplasmas in citrus worldwide.

Update and New Epidemiological Aspects about Grapevine Yellows in Chile.

To date, phytoplasmas belonging to six ribosomal subgroups have been detected to infect grapevines in Chile in 36 percent of the sampled plants. A new survey on the presence of grapevine yellows was carried out from 2016 to 2020, and 330 grapevine plants from the most important wine regions of the country were sampled and analyzed by nested PCR/RFLP analyses. Phytoplasmas enclosed in subgroups 16SrIII-J and 16SrVII-A were identified with infection rates of 17% and 2%, respectively. The vineyards in which the phytoplasma-infected plants were detected were further inspected to identify alternative host plants and insects of potential epidemiological relevance. Five previously unreported plant species resulted positive for 16SrIII-J phytoplasma (Rosa spp., Brassica rapa, Erodium spp., Malva spp. and Rubus ulmifolius) and five insect species were fully or partially identified (Amplicephalus ornatus, A. pallidus, A. curtulus, Bergallia sp., Exitianus obscurinervis) as potential vectors of 16SrIII-J phytoplasmas. The 16SrVII-A phytoplasmas were not detected in non-grape plant species nor in insects. This work establishes updated guidelines for the study, management, and prevention of grapevine yellows in Chile, and in other grapevine growing regions of South America.

Understanding Alstroemeria pallida Flower Colour: Links between Phenotype, Anthocyanins and Gene Expression.

Flower colour is mainly due to the accumulation of flavonoids, carotenoids and betalains in the petals. Of these pigments, flavonoids are responsible for a wide variety of colours ranging from pale yellow (flavones, flavonols and flavanodiols) to blue-violet (anthocyanins). This character plays a crucial ecological role by attracting and guiding pollinators. Moreover, in the ornamental plants market, colour has been consistently identified as the main feature chosen by consumers when buying flowers. Considering the importance of this character, the aim of this study was to evaluate flower colour in the native Chilean geophyte Alstroemeria pallida, by using three different approaches. Firstly, the phenotype was assessed using both a colour chart and a colourimeter, obtaining CIELab parameters. Secondly, the anthocyanin content of the pigmented tepals was evaluated by high-performance liquid chromatography (HPLC), and finally, the expression of two key flavonoid genes, chalcone synthase (CHS) and anthocyanidin synthase (ANS) was analysed using real-time polymerase chain reaction (PCR). Visual evaluation of A. pallida flower colour identified 5 accessions, ranging from white (Royal Horticultural Society (RHS) N999D) to pink (RHS 68C). Moreover, this visual evaluation of the accessions correlated highly with the CIELab parameters obtained by colourimetry. An anthocyanidin corresponding to a putative 6-hydroxycyanidin was identified, which was least abundant in the white accession (RHS N999D). Although CHS was not expressed differentially between the accessions, the expression of ANS was significantly higher in the accession with pink flowers (RHS 68C). These results suggest a correlation between phenotype, anthocyanin content and ANS expression for determining flower colour of A. pallida, which could be of interest for further studies, especially those related to the breeding of this species with ornamental value.

Draft Whole Genome Sequence Analyses on Pseudomonas syringae pv. actinidiae Hypersensitive Response Negative Strains Detected from Kiwifruit Bleeding Sap Samples.

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.

Draft Genome Sequence of 16SrIII-J Phytoplasma, a Plant Pathogenic Bacterium with a Broad Spectrum of Hosts.

Phytoplasmas are bacterial plant pathogens that can affect different vegetal hosts. In South America, a phytoplasma belonging to ribosomal subgroup 16SrIII-J has been reported in many crops. Here we report its genomic draft sequence, showing a total length of 687,253 bp and a G+C content of 27.72%.

Genetic divergence of tomato ringspot virus.

Tomato ringspot virus (ToRSV) has been detected in Chile, causing economically important diseases in a wide range of hosts. A ToRSV isolate was obtained from raspberry cv Heritage (Rasp-CL) showing leaf yellowing and stunting. The complete genome of Rasp-CL was sequenced by deep sequencing. The Rasp-CL RNA1 sequence shared 97.4 % nucleotide sequence identity with divergent RNA1 of isolate Rasp1-2014, while Rasp-CL RNA2 showed high divergence from all four isolates available in the database, sharing only 63.9-72.7 % nucleotide sequence identity. This difference was mainly based on the X4 coding region, which has been reported to be a high-variability region. Moreover, based on differences in the X4 region, three Rasp-CL RNA2 variants of different length were identified in the same host. One putative recombination event was identified between the Rasp-CL and GYV-2014 X4 genes. Phylogenetic analysis suggested that ToRSV isolates with currently available sequences form three distinct groups. Our results suggest that, for an accurate phylogenetic classification of ToRSV, it is necessary to obtain sequences of both RNAs. This is the first report of a complete ToRSV genome sequence from South America.

Simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus using real-time PCR and high resolution melting analysis.

In this study, the real-time PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed in a real-time PCR using a primer set for both of them or duplex real-time PCR that included one specific primer set for each virus. These two strategies allowed us to confirmed virus infection in all tested samples. In 17 field samples the technique revealed samples positive for CNRMV or CGRMV as well as positive for both viruses. In addition, the HRM analysis made it possible to differentiate clearly between CNRMV and CGRMV. Sequence variations among CNRMV and CGRMV isolates observed from the HRM peaks were confirmed by sequencing. To test the capability to use this method in field, forty one sweet cherry samples were examined by HRM analysis. The HRM data showed that seven samples were positive for CNRMV and three were infected with CGRMV. The results presented in this study indicated that real-time PCR followed by HRM analysis provides sensitive, automated and rapid tool to detect and differentiate between CNRMV and CGRMV isolates.

A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by prunus necrotic ringspot virus and peach latent mosaic viroid.

BACKGROUND: Microarray profiling is a powerful technique to investigate expression changes of large amounts of genes in response to specific environmental conditions. The majority of the studies investigating gene expression changes in virus-infected plants are limited to interactions between a virus and a model host plant, which usually is Arabidopsis thaliana or Nicotiana benthamiana. In the present work, we performed microarray profiling to explore changes in the expression profile of field-grown Prunus persica (peach) originating from Chile upon single and double infection with Prunus necrotic ringspot virus (PNRSV) and Peach latent mosaic viroid (PLMVd), worldwide natural pathogens of peach trees. RESULTS: Upon single PLMVd or PNRSV infection, the number of statistically significant gene expression changes was relatively low. By contrast, doubly-infected fruits presented a high number of differentially regulated genes. Among these, down-regulated genes were prevalent. Functional categorization of the gene expression changes upon double PLMVd and PNRSV infection revealed protein modification and degradation as the functional category with the highest percentage of repressed genes whereas induced genes encoded mainly proteins related to phosphate, C-compound and carbohydrate metabolism and also protein modification. Overrepresentation analysis upon double infection with PLMVd and PNRSV revealed specific functional categories over- and underrepresented among the repressed genes indicating active counter-defense mechanisms of the pathogens during infection. CONCLUSIONS: Our results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits. We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections. Thus, our data demonstrate for the first time that the simultaneous infection of a viroid and a plant virus synergistically affect the host transcriptome in infected peach fruits. These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.