Sweet Cherry Plants Prioritize Their Response to Cope with Summer Drought, Overshadowing the Defense Response to Pseudomonas syringae pv. syringae.

Disease severity and drought due to climate change present significant challenges to orchard productivity. This study examines the effects of spring inoculation with Pseudomonas syringae pv. syringae (Pss) on sweet cherry plants, cvs. Bing and Santina with varying defense responses, assessing plant growth, physiological variables (water potential, gas exchange, and plant hydraulic conductance), and the levels of abscisic acid (ABA) and salicylic acid (SA) under two summer irrigation levels. Pss inoculation elicited a more pronounced response in 'Santina' compared to 'Bing' at 14 days post-inoculation (dpi), and those plants inoculated with Pss exhibited a slower leaf growth and reduced transpiration compared to control plants during 60 dpi. During differential irrigations, leaf area was reduced 14% and 44% in Pss inoculated plants of 'Bing' and 'Santina' respectively, under well-watered (WW) conditions, without changes in plant water status or gas exchange. Conversely, water-deficit (WD) conditions led to gas exchange limitations and a 43% decrease in plant biomass compared to that under WW conditions, with no differences between inoculation treatments. ABA levels were lower under WW than under WD at 90 dpi, while SA levels were significantly higher in Pss-inoculated plants under WW conditions. These findings underscore the influence on plant growth during summer in sweet cherry cultivars that showed a differential response to Pss inoculations and how the relationship between ABA and SA changes in plant drought level responses.

Phytoplasma-Associated Diseases in South America: Thirty Years of Research.

Phytoplasma-associated diseases are mainly insect-transmitted and are present worldwide. Considering that disease detection is a relevant environmental factor that may elucidate the presence of these diseases, a review reporting the geographic distribution of phytoplasma taxa in geographically consistent areas helps manage diseases appropriately and reduce their spreading. This work summarizes the data available about the identification of the phytoplasma associated with several diverse diseases in South America in the last decades. The insect vectors and putative vectors together with the plant host range of these phytoplasmas are also summarized. Overall, 16 'Candidatus Phytoplasma' species were detected, and those most frequently detected in agricultural-relevant crops such as corn, alfalfa, grapevine, and other horticultural species are 'Ca. P. pruni', 'Ca. P. asteris', and 'Ca. P. fraxini'.

Nitric Oxide Mitigates the Deleterious Effects Caused by Infection of Pseudomonas syringae pv. syringae and Modulates the Carbon Assimilation Process in Sweet Cherry under Water Stress.

Bacterial canker is an important disease of sweet cherry plants mainly caused by Pseudomonas syringae pv. syringae (Pss). Water deficit profoundly impairs the yield of this crop. Nitric oxide (NO) is a molecule that plays an important role in the plant defense mechanisms. To evaluate the protection exerted by NO against Pss infection under normal or water-restricted conditions, sodium nitroprusside (SNP), a NO donor, was applied to sweet cherry plants cv. Lapins, before they were exposed to Pss infection under normal or water-restricted conditions throughout two seasons. Well-watered plants treated with exogenous NO presented a lower susceptibility to Pss. A lower susceptibility to Pss was also induced in plants by water stress and this effect was increased when water stress was accompanied by exogenous NO. The lower susceptibility to Pss induced either by exogenous NO or water stress was accompanied by a decrease in the internal bacterial population. In well-watered plants, exogenous NO increased the stomatal conductance and the net CO2 assimilation. In water-stressed plants, NO induced an increase in the leaf membranes stability and proline content, but not an increase in the CO2 assimilation or the stomatal conductance.

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 that is 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.

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.

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.

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.

A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.

At least 58 viruses have been reported to infect grapevines causing economic damage globally. Conventional detection strategies based on serological assays, biological indexing and RT-PCR targeting one or few viruses in each assay are widely used. Grapevines are prone to contain mixed infections of several viruses, making the use of these techniques time-consuming. A 70-mer oligonucleotide microarray able to detect simultaneously a broad spectrum of known viruses as well as new viruses by cross-hybridization to highly conserved probes is reported in the present study. The array contains 570 unique probes designed against highly conserved and species-specific regions of 44 plant viral genomes. In addition probes designed against plant housekeeping genes are also included. By using a random primed RT-PCR amplification strategy of grapevine double stranded RNA-enriched samples, viral agents were detected in single and mixed infections. The microarray accuracy to detect 10 grapevine viruses was compared with RT-PCR yielding consistent results. For this purpose, grapevine samples containing single or mixed infections of Grapevine leafroll-associated virus-1, -2, -3, -4, -7, -9, Grapevine fanleaf virus, Grapevine rupestris stem pitting-associated virus, Grapevine virus A, and Grapevine virus B were used. Genomic libraries containing complete viral genomes were also used as part of the validation process. The specific probe hybridization pattern obtained from each virus makes this approach a powerful tool for high throughput plant certification purposes and also for virus discovery if the new viral genomic sequences have partial similarity with the microarray probes. Three Closteroviridae members (Grapevine leafroll-associated virus -4, -7 and -9) were detected for the first time in Chilean grapevines using the microarray.

Genetic diversity of the movement and coat protein genes of South American isolates of Prunus necrotic ringspot virus.

Prunus necrotic ringspot virus (PNRSV) is distributed worldwide, but no molecular data have been previously reported from South American isolates. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of 23 isolates of PNRSV from Chile, Brazil, and Uruguay, and from different Prunus species, have been obtained. Phylogenetic analysis performed with full-length MP and CP sequences from all the PNRSV isolates confirmed the clustering of the isolates into the previously reported PV32-I, PV96-II and PE5-III phylogroups. No association was found between specific sequences and host, geographic origin or symptomatology. Comparative analysis showed that both MP and CP have phylogroup-specific amino acids and all of the motifs previously characterized for both proteins. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that most amino acid sites are under the effect of negative purifying selection.