First Report of Erwinia aphidicola Causing Bulb Rot of Onion in Chile.

During the 2021-22 and 2022-23 seasons (December to February), onion plants (Allium cepa L.) showing decay, leaf blight, chlorosis and water soak lesions were collected in Central Chile. Five symptomatic plants were sampled from 20 different onion fields. Brown rot of the external scales was observed in bulbs from two fields: one planted with the cv. Campero (20 ha; O'Higgins Region), and another with cv. Marenge (2 ha; Metropolitan Region). The disease incidence in these fields ranged from 2% to 5%. Isolations were carried out from symptomatic leaves and bulbs from these fields on King's B medium, resulting in small white colonies with smooth margin. Three isolates were selected, two from first field (QCJ3A & QCJ2B), and one from second field (EPB1). A preliminary identification based on 16S rRNA sequences was conducted. BLAST analyses of strains QCJ3A, QCJ2B and EPB1 (GenBank Accession No. PP345601 to PP345603) against the NCBI Database resulted in a match with strains (GenBank Accession No. ON255770.1 and ON255825.1) isolated from infected bulbs in Texas, USA identified as Erwinia spp. (Khanal et al. 2023), with 100% coverage and 100% identity (707 bp out of 707). To evaluate the pathogenicity of these three strains, onion bulbs were inoculated (Guajardo et al. 2023). Toothpicks previously immersed in a bacterial suspension at ~ 108 colony forming units (CFU)/mL were pricked at a 4 cm depth into the shoulders of onion bulbs bought from commercial store and incubated at room temperature. Bulbs inoculated with sterile water served as negative control. A known onion bulb rotting bacterial strain of Dickeya sp. was used as a positive control. At the end of the incubation period (20 days), bulbs were opened longitudinally across their inoculation site, showing that the external scales had a brown color. Negative control remained asymptomatic. Strains were re-isolated from damaged tissue and identified as Erwinia sp. This assay was repeated three times with the same results. For further identification, genomic DNA extraction was carried out using the Blood & Cell Culture DNA Kit (Qiagen), and genome sequencing was performed in the Illumina HiSeq 2500 platform. The Whole Genome Shotgun project for strains QCJ3A, QCJ2B and EPB1 have been deposited at DDBJ/ENA/GenBank under the accession JBANEI010000000, JBANEJ010000000 and JBANEK010000000. The average nucleotide identity (ANI) values were 99.6% (EPB1), 98.2% (QCJ2B), and 99.6% (QCJ3A) and DNA-DNA hybridization (dDDH) values were 96.9% (EPB1), 83.7% (QCJ2B), and 97.1% (QCJ3A), when compared with the type strain Erwinia aphidicola JCM 21238 (GenBank accession No. GCF_014773485.1). The three strains were deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM). Erwinia aphidicola has been previously described causing diseases in common bean (Phaseolus vulgaris) and pea (Pisum sativum), in Spain (Santos et al. 2009) and in pepper (Capsicum annuum) in China (Luo et al. 2018). Its close relative E. persicina has been reported causing bulb rot in onion in Korea (Cho et al. 2019) and garlic in Europe (Galvez et al. 2015). To our knowledge, this is the first report of E. aphidicola causing a bulb rot of onion in Chile. Although the distribution and prevalence of this bacterium in Chilean agroecosystems is not known, it can be a potential cause of losses in onions and other crops such as beans, peas, and peppers. Additional studies should be conducted to determine the host range of Chilean Erwinia aphidicola strains.

First Report of Pantoea ananatis and Pantoea eucalypti causing onion leaf blight and bulb decay in Central Chile.

Nearly 5,400 hectares of long-day onions (Allium cepa) are cultivated in the Central Zone of Chile (UTM 33°3´S to 37°24´S). During summer 2021-22 and 2022-23 (December to February), followed by high temperatures (around 35°C) and high humidity conditions, symptoms showing yellowing, soft rot, blight on old leaves, necrotic tips, and soft bulbs were observed. The affected plants were observed in "spots" into the fields, and the incidence reached 5-10%. The severity was high, and 70-80% of the affected plants died. Symptomatic plants from different fields from the Coquimbo, Metropolitan, O´Higgins and Maule regions were sampled. Isolations were made using casamino-acid peptone glucose (CPG) agar medium (Schaad, 2001). Yellow-pigmented, circular to irregular shaped colonies were observed. Molecular identification was carried out by partial 16S rRNA gene sequencing, resulting in the identification of 18 isolates of Pantoea spp. from twelve different fields. Selected strains were biochemically analyzed using the GEN III BIOLOG microtest system (Hayward, CA) and were identified as Pantoea spp. BLAST analyses of the 16S rRNA sequences (602 nt) of selected strains (GenBank Accession No. OR527817 to OR527819) against the NCBI Database resulted in the identification of Pantoea species with 100% coverage and 100% identity. To determine the Pantoea species of each strain, housekeeping gene gyrB (Delétoile et al. 2009) was amplified and sequenced (GenBank Accessions No. OR544061 to OR544063). BLAST analysis (802 nt) of selected strains resulted in 100% coverage and 100% identity, identifying three different species: P. ananatis, P. eucalypti, and P. agglomerans. Pantoea species were isolated from both leaves and bulbs and no more than one species was observed per field. Pathogenicity assays in onion plants and bulbs were performed based on the methodology described by Asselin et al. (2018). Five onion plants cv. Cimarron of thirteen-week-old were inoculated by wounding an external leave with a sterile toothpick previously immersed in a bacterial suspension at ~ 108 CFU/mL and maintained at 26-28°C for 30 days in high humidity conditions. Control plants were inoculated with sterile water. Plants inoculated with Pantoea spp. showed chlorosis, soft rot, and necrosis mainly in older leaves, as observed in the field, while negative control plants remained healthy. Pantoea spp. were re-isolated from the inoculated onion plants. Toothpicks dipped in the inoculum were stuck 4 cm into the shoulders of onion bulbs and incubated at 26°C for 20 days. Water was inoculated as a negative control. At the end of the incubation period, the bulbs were opened longitudinally across their inoculation sites and shrunken, brownish, watery scales were observed. Pantoea species have been previously described as causing leaf blight of onions in Georgia and Michigan in the USA and South Africa, Brazil, and Uruguay (Hattingh and Walters 1981; Gitaitis and Gay 1997; Edens et al. 2006; Tho et al. 2015; De Armas et al. 2022; Rosende et al. 2022). P. agglomerans was recently reported in Chile (Sepúlveda et al. 2023), but this is the first report of P. ananatis and P. eucalypti affecting onions in central Chile. This detection is an alert call for the onion's growers and exporters in Chile for upcoming seasons, where conditions predisposing to disease may continue to occur. It is crucial to continue analyzing the factors that caused the appearance of this new disease in onions.

A Comparison of Immediate and Short-Term Defensive Responses to Phytophthora Species Infection in Both Susceptible and Resistant Walnut Rootstocks.

Clonal rootstocks are one alternative used by the walnut industry to control damage caused by Phytophthora species, traditionally using plants grafted on susceptible Juglans regia rootstock. Vlach, VX211, and RX1 are clonal rootstocks with a degree of resistance to Phytophthora species. The resistance to pathogens in these rootstocks depends on the resistance mechanisms activated by the presence of the pathogen and subsequent development of responses in the host. In this work, we analyzed how plants of J. regia, Vlach, VX211, and RX1 responded to inoculation with Phytophthora cinnamomi or Phytophthora citrophthora isolates obtained from diseased English walnut plants from Chilean orchards. After inoculation, plants of Vlach, VX211, and RX1 showed canopy and root damage indexes that did not differ from noninoculated control plants. In contrast, plants of J. regia, which is susceptible to P. cinnamomi and P. citrophthora, died after inoculation. Vlach, VX211, and RX1 plants inoculated with P. cinnamomi or P. citrophthora showed greater root weight and volume and greater root growth rates than their respective controls. These results suggest that short-term carbohydrate dynamics may be related to the defense mechanisms of plants; they are immediately activated after inoculation through the production of phenolic compounds, which support the further growth and development of roots in walnut clonal rootstocks. To our knowledge, this is the first study that comprehensively characterizes vegetative and radicular growth and the dynamics of sugars and phenols in response to infection with P. cinnamomi or P. citrophthora in walnut rootstocks.

Characterization of Oomycete Species Associated With Root and Crown Rot of English Walnut in Chile.

English (Persian) walnut (Juglans regia) trees affected by root and crown rot were surveyed in five regions of central Chile between 2015 and 2017. In each region, nine orchards, ranging from 1 to 21 years old, were randomly selected and inspected for incidence and severity of tree decline associated with crown and root rot. Soil and symptomatic crown and root tissues were collected and cultured in P5ARP semiselective medium to isolate potential oomycete pathogens, which were identified through morphology and molecularly using ITS sequences in the rDNA gene and beta tubulin gene. The most frequently isolated species was Phytophthora cinnamomi. Pathogenicity tests were conducted with representative oomycete isolates. P. cinnamomi, P. citrophthora, and Pythium ultimum were all pathogenic in J. regia. Nevertheless, only P. cinnamomi and P. citrophthora were pathogenic to English walnut. Py. ultimum caused limited levels of root damage to English walnut seedlings. Our research indicates that as the Chilean walnut industry has expanded, so have walnut crown and root rots induced by oomycetes.