A New Multiplex TaqMan qPCR for Precise Detection and Quantification of Clavibacter michiganensis in Seeds and Plant Tissue.

Bacterial canker of tomato caused by Clavibacter michiganensis (Cm) is one of the most devastating bacterial diseases affecting the tomato industry worldwide. As the result of Cm colonization of the xylem, the susceptible host shows typical symptoms of wilt, marginal leaf necrosis, stem cankers, and ultimately plant death. However, is the ability of Cm to infect seeds and plants without causing symptoms what makes it an even more dangerous pathogen. Unfortunately, there are no resistant cultivars or effective chemical or biological control methods available to growers against Cm. Its control relies heavily on prevention. The implementation of a rapid and accurate detection tool is imperative to monitor the presence of Cm and prevent its spread. In this study, we developed a specific and sensitive multiplex TaqMan qPCR assay to detect Cm and distinguish it from related bacterial species that affect tomato plants. Two Cm chromosomal virulence-related genes, rhuM and tomA, were used as specific targets. The plant internal control tubulin alpha-3 was included in each of the multiplexes to improve the reliability of the assay. Specificity was evaluated with 37 bacterial strains including other Clavibacter spp. and related and unrelated bacterial pathogens from different geographic locations affecting a wide variety of hosts. Results showed that the assay is able to discriminate Cm strains from other related bacteria. The assay was validated on tissue and seed samples following artificial infection and all tested samples accurately detected the presence of Cm. The tool described here is highly specific, sensitive, and reliable for the detection of Cm and allows the quantification of Cm in seeds, roots, stems, and leaves, and roots. The diagnostic assay can also be adapted for multiple purposes such as seed certification programs, surveillance, biosafety, the effectiveness of control methods, border protection, and epidemiological studies.

Occurrence of Pseudomonas cichorii causing midrib rot of lettuce under hydroponic cultivation in Chile.

In Chile, lettuce is the vegetable that has increased in cultivated area the most in recent years, reaching 8,309 ha. The Coquimbo Region contributes the most to this growth in production with 3,284 ha in 2022 (ODEPA 2023). Most lettuce is grown under open field conditions, but there is significant production in greenhouses and an increase in hydroponic production systems (INIA 2017). During April to June 2021 and 2022 in the Coquimbo Region, butterhead-type lettuce seedlings (Lactuca sativa) cv. Neil, cultivated under a hydroponic system, showed severe brown to black lesions in the leaves and midrib (Figure S1). To determine the etiology of this problem, samples of diseased plants were taken. Pieces of symptomatic tissue were macerated, and the extract was spread on nutrient agar (NA) and on King's B medium (KB) and incubated at 23°C for 48 h. The bacterial colonies observed were predominantly circular, creamy-white in color with irregular margins and fluorescent in KB medium. Isolates were gram-negative strictly aerobic. LOPAT test (Lelliot et al. 1966) results of two selected isolates were: levan production (-), oxidase reaction (+), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+), which corresponds to the profile of Pseudomonas cichorii. Molecular identification was performed through amplification and sequencing of the 16S rRNA (GenBank Accessions No. OR540674 to OR540675), gyrB and rpoD genes (Hwang et al. 2005; Sarkar and Guttmann 2004) (GenBank Accessions No. OR558279 to OR558282). BLAST analysis of the 16S rRNA gene of the isolates resulted in a match with a 99.86% identity with P. cichorii type strain ATCC 10857 (NR_112070.1). BLAST analysis of gyrB and rpoD resulted in a match with a 100% (630/630 bp) and >99% (546/550 bp) identity respectively, with strains of P. cichorii. Five six-month-old lettuce plants cv. Desert Storm were pricked in the midrib with a toothpick smeared with a fresh colony grown on KB medium. Seven days after inoculation, the plants showed dark brown, watery lesions, characteristic of damage caused by P. cichorii (Figure S1). Bacteria were isolated again from the inoculated plants and were identified as P. cichorii using LOPAT and molecular identification techniques. Midrib rot caused by P. cichorii was reported as an emerging disease of greenhouse-grown lettuce by Cottyn et al. (2009). In Chile, P. cichorii was previously described affecting nectarine fruits (Pinto de Torres and Carreño Ibañez 1983) and reported as a pathogen of lettuce among others horticultural crops by Servicio Agrícola y Ganadero of the Government of Chile (Acuña 2008), but this is the first report of P. cichorii affecting hydroponic lettuce plants in Chile. These results will be the basis of future studies to evaluate the origin of the infection, the potential dissemination, and the implementation of disease management to avoid the damage caused by this bacterium in hydroponic systems in this crop of growing importance in Chile.

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.

Rootstock increases the physiological defence of tomato plants against Pseudomonas syringae pv. tomato infection.

Climate change has intensified the infection of tomato plants by pathogens such as Pseudomonas syringae pv. tomato (Pst). Rootstocks may increase plant tolerance to leaf phytopathogens. The aim of this study was to evaluate the effects of the tolerant Poncho Negro (R) tomato rootstock on physiological defence and the role of hydrogen sulfide (H2S) in susceptible Limachino (L) tomato plant responses to Pst attack. Ungrafted (L), self-grafted (L/L), and grafted (L/R) plants were infected with Pst. Rootstock increased the concentration of antioxidant compounds including ascorbate in the scion. Tolerant rootstock induced an increase of H2S in the scion, which correlated with enhanced expression of the SlAPX2 gene. A high accumulation of salicylic acid was observed in Pst-inoculated grafted L/L and L/R plants, but this was higher in L/R plants. The increase of H2S during Pst infection was associated with a reduction of ethylene in L/R plants. Our study indicates that the Poncho Negro rootstock reduced the symptoms of bacterial speck disease in the Limachino tomato plants, conferring tolerance to Pst infection. This study provides new knowledge about the impact of rootstock in the defence of tomato plants against leaf pathogens that could be used in sustainable management of tomato cultivation.

First Report of bacterial speck caused by Pseudomonas syringae pv. tomato Race 1 affecting tomato in different Regions of Chile.

In Chile, tomato is one of the most widely cultivated vegetables, with around 5,000 ha for fresh market and 8,000 ha for processing industry. During recent years, symptoms of bacterial speck caused by Pseudomonas syringae pv. tomato, have been observed more frequently in tomato plants in different regions of Chile. This pathogen was first identified in Chile in 1987 (Latorre & Lolas, 1988) and the presence of an apparent new variant was reported in 2004 (Besoain et al. 2004). To characterize the pathogen that was affecting this crop, samples of diseased tomato plants were taken in three regions of Chile. The samples were collected in 2016 in Northern Chile in Lluta Valley from the Arica y Parinacota Region, and in Central Chile, in 2014 in Limache from Valparaíso Region and in 2015 in Pichidegua from O´Higgins Region. Affected tomato plants exhibited dark brown to black lesions surrounded by yellow halos in the leaves, and dark brown to black lesions in the stems, pedicels, and peduncles. Plants tissues were macerated, and the suspension was spread on King's B medium, resulting in fluorescent colonies visualized under 366 nm UV light. LOPAT tests results of three selected isolates from different Regions, were: levan production (+), oxidase reaction (-), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+) (Lelliot et al. 1966). Molecular identification was carried out by amplification and sequence analysis of housekeeping genes cts, encoding citrate synthase, gyrB, encoding DNA gyrase B, and rpoD, encoding sigma factor 70 (Hwang et al. 2005; Sarkar & Guttmann 2004) (GenBank Accessions No. OK001658-OK001666). BLAST analysis of cts and rpoD genes of the three isolates resulted in a match with a 100% identity (919 bp and 491 bp respectively) with Pseudomonas syringae pv. tomato strain B13-200 (GenBank: CP019871.1). BLAST analysis of gyrB gene of two isolates resulted in a match with a 100% identity (684 bp) and one isolate with 99.85% (683 bp) with Pseudomonas syringae pv. tomato strain B13-200. To identify the race 1, each strain was inoculated in five tomato plants cv. San Pedro, susceptible to both races of P. syringae pv. tomato, and cv. Rio Grande, resistant to race 0. The tomato plants were slightly wounded with a metal sponge and then sprayed with the bacterial suspension (108 CFU mL-1) of each isolate, including the reference strain DC3000 (race 0). Negative controls were sprayed with water. The plants inoculated with Chilean strains in both cv. San Pedro and cv. Rio Grande, showed symptoms of bacterial speck after 7 days. Plants inoculated with DC3000 strain showed symptoms only in cv. San Pedro, whereas control plants remained asymptomatic. Strains were re-isolated from symptomatic plants and identified by gene sequence analyses as Pseudomonas syryngae pv. tomato. This is the first report of Pseudomonas syryngae pv. tomato race 1 in Chile. Race 1 was previously reported in Canada (Lawton and MacNeill. 1986), in Italy (Buonaurio et al. 1996), in California (Arredondo and Davis 2000), in Portugal (Cruz et al. 2010), and in other states in the USA and countries in South America, Europe, Africa, and Australia, becoming the most commonly isolated race today (Cai et al 2011). These results will be the base for future studies of epidemiology, characterization, and virulence in order to explain the outbreak of this disease and the severity of symptoms observed.

Antifungal Nanoformulation for Biocontrol of Tomato Root and Crown Rot Caused by Fusarium oxysporum f. sp. radicis-lycopersici.

Tomatoes (Solanum lycopersicum L.) are the most cultivated and important vegetable crop in the world. These plants can wilt during crop growth due to fusarium wilt (fusariosis), a disease that damages tomato vascular systems. The Fusarium isolated and analyzed in this work correspond to Fusarium oxysporum f. sp. radicis-lycopersici. The isolates were molecularly identified, and analysis was done on the in vitro effects of the nanoemulsions (previously obtained from extracts of Chilean medicinal plants of the genera Psoralea and Escallonia) to inhibit mycelial and conidial germination of the isolates. Subsequently, the nanoemulsions were evaluated under greenhouse conditions for preventive control of fusariosis in the root and crown, with high levels of disease control observed using the highest concentrations of these nanoemulsions, at 250 and 500 ppm.

Analyses of Virulence Genes of Clavibacter michiganensis subsp. michiganensis Strains Reveal Heterogeneity and Deletions That Correlate with Pathogenicity.

Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial canker of tomato. Differences in virulence between Cmm strains have been reported. The aim of this study was the characterization of nine Cmm strains isolated in Chile to reveal the causes of their differences in virulence. The virulence assays in tomato seedlings revealed different levels of severity associated with the strains, with two highly virulent strains and one causing only mild symptoms. The two most virulent showed increased cellulase activity, and no cellulase activity was observed in the strain causing mild symptoms. In three strains, including the two most virulent strains, PCR amplification of the 10 virulence genes analyzed was observed. In the strain causing mild symptoms, no amplification was observed for five genes, including celA. Sequence and cluster analyses of six virulence genes grouped the strains, as has been previously reported, except for gene pelA1. Gene sequence analysis from the genomes of five Chilean strains revealed the presence of deletions in the virulence genes, celB, xysA, pat-1, and phpA. The results of this study allow us to establish correlations between the differences observed in disease severity and the presence/absence of genes and deletions not previously reported.

Microbial Diversity of Psychrotolerant Bacteria Isolated from Wild Flora of Andes Mountains and Patagonia of Chile towards the Selection of Plant Growth-Promoting Bacterial Consortia to Alleviate Cold Stress in Plants.

Cold stress decreases the growth and productivity of agricultural crops. Psychrotolerant plant growth-promoting bacteria (PGPB) may protect and promote plant growth at low temperatures. The aims of this study were to isolate and characterize psychrotolerant PGPB from wild flora of Andes Mountains and Patagonia of Chile and to formulate PGPB consortia. Psychrotolerant strains were isolated from 11 wild plants (rhizosphere and phyllosphere) during winter of 2015. For the first time, bacteria associated with Calycera, Orites, and Chusquea plant genera were reported. More than 50% of the 130 isolates showed ≥33% bacterial cell survival at temperatures below zero. Seventy strains of Pseudomonas, Curtobacterium, Janthinobacterium, Stenotrophomonas, Serratia, Brevundimonas, Xanthomonas, Frondihabitans, Arthrobacter, Pseudarthrobacter, Paenarthrobacter, Brachybacterium, Clavibacter, Sporosarcina, Bacillus, Solibacillus, Flavobacterium, and Pedobacter genera were identified by 16S rRNA gene sequence analyses. Ten strains were selected based on psychrotolerance, auxin production, phosphate solubilization, presence of nifH (nitrogenase reductase) and acdS (1-aminocyclopropane-1-carboxylate (ACC) deaminase) genes, and anti-phytopathogenic activities. Two of the three bacterial consortia formulated promoted tomato plant growth under normal and cold stress conditions. The bacterial consortium composed of Pseudomonas sp. TmR5a & Curtobacterium sp. BmP22c that possesses ACC deaminase and ice recrystallization inhibition activities is a promising candidate for future cold stress studies.

Activity of Adesmia boronioides resinous exudate against phytopathogenic bacteria.

Resinous exudate obtained from the aerial parts of Adesmia boronioides Hook.f. were evaluated to determine anti-phytopathogenic effects. Briefly, resinous exudate was obtained by dipping fresh plant material in dichloromethane; chemical composition was determined by GC-MS; and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated against four phytopathogenic bacteria. Resinous exudate yield was 8.5% (resin/fresh plant), of which esquel-6-en-9-one (14.25%), esquel-7-en-9-one (5.86%), and veratric acid (2.59%) were the effective antibacterial compounds. Tested against Pectobacterium carotovorum subsp. carotovora, Erwinia amylovora, Bacillus subtilis, and Pseudomonas syringae, MICs and MBCs ranged from 16 to 128 µg/mL and 32-256 µg/mL, respectively. These results provide initial evidence that resinous bush A. boronioides is a new and alternative source of substances with agricultural interest.

Comparative Genomics of Pathogenic Clavibacter michiganensis subsp. michiganensis Strains from Chile Reveals Potential Virulence Features for Tomato Plants.

The genus Clavibacter has been associated largely with plant diseases. The aims of this study were to characterize the genomes and the virulence factors of Chilean C. michiganensis subsp. michiganensis strains VL527, MSF322 and OP3, and to define their phylogenomic positions within the species, Clavibacter michiganensis. VL527 and MSF322 genomes possess 3,396,632 and 3,399,199 bp, respectively, with a pCM2-like plasmid in strain VL527, with pCM1- and pCM2-like plasmids in strain MSF322. OP3 genome is composed of a chromosome and three plasmids (including pCM1- and pCM2-like plasmids) of 3,466,104 bp. Genomic analyses confirmed the phylogenetic relationships of the Chilean strains among C.michiganensis subsp. michiganensis and showed their low genomic diversity. Different virulence levels in tomato plants were observable. Phylogenetic analyses of the virulence factors revealed that the pelA1 gene (chp/tomA region)-that grouped Chilean strains in three distinct clusters-and proteases and hydrolases encoding genes, exclusive for each of the Chilean strains, may be involved in these observed virulence levels. Based on genomic similarity (ANIm) analyses, a proposal to combine and reclassify C. michiganensis subsp. phaseoli and subsp. chilensis at the species level, as C. phaseoli sp. nov., as well as to reclassify C. michiganensis subsp. californiensis as the species C. californiensis sp. nov. may be justified.

The arbuscular mycorrhizal fungus Funneliformis mosseae induces changes and increases the concentration of volatile organic compounds in Vitis vinifera cv. Sangiovese leaf tissue.

Arbuscular mycorrhizal fungi (AMF) are beneficial obligate symbionts of plant roots. Volatile organic compounds (VOCs) participate in plant communication and defence. The aim of this study was to analyse the effects of the arbuscular mycorrhizal fungus Funneliformis mosseae IMA1 on VOCs in Vitis vinifera cv. Sangiovese leaf tissue. Grapevine plants inoculated with F. mosseae IMA1 were incubated for 23 weeks. VOCs were extracted from leaves and identified using headspace solid-phase microextraction (HS-SPME) coupled to GC-MS. VOCs in leaf tissue were strongly enhanced (85%) by F. mosseae IMA1. The mycorrhizal fungus IMA1 modified the levels of specific VOCs synthesised in different anabolic pathways. An increase in volatiles that have been related to plant defences under pathogen/herbivore attack or linked to water stress, such as (E)-2-hexenal, 3-hexenal, geraniol, benzaldehyde and methyl salicylate, was observed in mycorrhizal plants. In contrast, some C13-norisoprenoids decreased strongly in mycorrhizal plants. The study of the effects of AMF on VOCs in grapevine plants may provide useful information to establish sustainable viticultural practices.

Multiple Introductions of Tomato Pathogen Clavibacter michiganensis subsp. michiganensis into Iran as Revealed by a Global-Scale Phylogeographic Analysis.

Tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is one of the most important seed-borne tomato diseases around the globe. The disease was initially reported in 1993 in Iran, and it became a rising threat for the multibillion dollar tomato industry of the country during the last decade. In this study, using phylogeographic analyses, we determined genetic diversity and geographic distribution of C. michiganensis subsp. michiganensis in Iran. Our field surveys showed that the pathogen is expanding into the southern and eastern areas of the country. Furthermore, multilocus sequence analysis and typing (MLSA/MLST) using the sequences of five housekeeping genes (atpD, gyrB, ppk, recA, and rpoB) revealed that 37 C. michiganensis subsp. michiganensis strains isolated in Iran had high genetic diversity and placed in 15 sequence types (STs), while all the available 184 worldwide C. michiganensis subsp. michiganensis sequences were placed in 43 STs. MLSA divided the worldwide C. michiganensis subsp. michiganensis strains into two phylogroups (I and II). Among the 37 strains isolated in Iran, 30 strains clustered in phylogroup I, while 7 strains clustered in phylogroup II. Phylogeographic data inferred from the allelic profile of the five housekeeping genes suggested multiple introductions of C. michiganensis subsp. michiganensis inoculum into Iran, while the geographic origin of the Iranian C. michiganensis subsp. michiganensis strains remains undetermined. Further analyses using higher numbers of strains are warranted to decipher the evolutionary history of C. michiganensis subsp. michiganensis in Iran. Additionally, stricter seed/transplant inspections are recommended to reduce the risk of pathogen expansion to areas with no history of the disease.IMPORTANCEClavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker disease, is one of the economically important pathogens of solanaceous crops (e.g., eggplant, pepper, and tomato) around the world. The disease occurs in many countries, with a particular importance in regions characterized by high precipitation and humid environmental conditions. As a seed-borne pathogen, C. michiganensis subsp. michiganensis is included in the A2 (high risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Bacterial canker disease was reported for the first time in 1993 in Iran, while the geographic distribution, genetic diversity, and phylogenetic position of the causal agent remain undetermined. In this study, using the multilocus sequence analysis and typing (MLSA/MLST) approach, we provided a phylogeographic scheme for the C. michiganensis subsp. michiganensis strains isolated in Iran. Furthermore, global-scale phylogenetic analyses led to determination of phylogenetic position of Iranian C. michiganensis subsp. michiganensis strains among worldwide population of the pathogen. Based on diversity parameters and population structure, we suggest relatively higher genetic diversity of the bacterial canker pathogen in Iran than has so far been observed in the other areas of the world. Results obtained in this study provide a novel insight into the genetic diversity and population structure of the bacterial canker pathogen on a global scale.

Biopesticide Activity from Drimanic Compounds to Control Tomato Pathogens.

Tomato crops can be affected by several infectious diseases produced by bacteria, fungi, and oomycetes. Four phytopathogens are of special concern because of the major economic losses they generate worldwide in tomato production; Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato, causative agents behind two highly destructive diseases, bacterial canker and bacterial speck, respectively; fungus Fusarium oxysporum f. sp. lycopersici that causes Fusarium Wilt, which strongly affects tomato crops; and finally, Phytophthora spp., which affect both potato and tomato crops. Polygodial (1), drimenol (2), isonordrimenone (3), and nordrimenone (4) were studied against these four phytopathogenic microorganisms. Among them, compound 1, obtained from Drimys winteri Forst, and synthetic compound 4 are shown here to have potent activity. Most promisingly, the results showed that compounds 1 and 4 affect Clavibacter michiganensis growth at minimal inhibitory concentrations (MIC) values of 16 and 32 µg/mL, respectively, and high antimycotic activity against Fusarium oxysporum and Phytophthora spp. with MIC of 64 µg/mL. The results of the present study suggest novel treatment alternatives with drimane compounds against bacterial and fungal plant pathogens.