First Report of a Leaf Spot Disease of Tobacco Caused by Pseudomonas cichorii in China

Tobacco (Nicotiana tabacum L.), one of the chief commercial crops, is wildly cultivated worldwide. In June 2020 and 2021, an unknown bacterial leaf spot on tobacco was found in Hezhou and Hechi City, Guangxi, China. 30% of the tobacco were affected and the rate of diseased leaves reached about 10% in the field under high temperature and rainstorm. The disease mainly damaged the middle and top leaves of tobacco plants at vigorous growing stage. The initial symptoms were water-soaked spots on the frontal half of a leaf, and then expanded into circular to irregular spots with a yellow halo at the edge. The spots mostly appeared dark brown at high air humidity, while yellow brown at low humidity and exhibited a concentric pattern. In severe cases, the lesions coalesced and the whole leaf was densely covered with lesions, resulting in the loss of baking value. A bacterium was consistently isolated from diseased leaf tissues on nutrient agar (NA). Growth on NA was predominantly grayish white circular bacterial colonies with smooth margins, and the bacterium is rod-shaped, gram-negative and fluorescent on King’s B medium. Seven isolates (ND04A-ND04C and ZSXF02-ZSXF05) were selected for molecular identification and pathogenicity tests. Genomic DNA of the bacterium was extracted and the housekeeping gene of cts (encoding citrate synthase) was amplified with the primers cts-Fs/cts-Rs (forward primer cts-Fs: 5’-CCCGTCGAGCTGCCAATWCTGA-3’; reverse primer cts-Rs: 5’-ATCTCGCACGGSGTRTTGAACATC-3’) (Berge et al. 2014; Sarkar et al. 2004). 409-bp cts gene sequences were deposited in the GenBank database for seven isolates (accession no. OK105110-OK105116). Sequence of seven isolates shared 100% identity with several Pseudomonas cichorii strains within the GenBank database (accession no. KY940268 and KY940271), and the phylogenetic tree of cts genes of the seven isolates clustered with the phylogroup 11 of Pseudomonas syringae (accession no. KJ877799 and KJ878111), which was classified as P.cichorii. To satisfy Koch’s postulates, a pathogenicity test was tested by using a needle to dip a suspension of the bacterium (108 CFU/ml) and pricking three holes in the tobacco leaf. The control plants leaves were needled with sterile water. Each tobacco plant was inoculated with three leaves, and the test was repeated three times. All plants were placed in transparent plastic boxes and incubated in a greenhouse at 25 ± 3°C. The water-soaked spots appeared 24h after inoculation and quickly expanded through leaf veins. Three days after inoculation, all the inoculated leaves showed symptoms similar to those observed in the field. Control plants remained healthy. Only P. cichorii was successfully re-isolated from the lesions, confirming Koch’s postulates. Pseudomonas cichorii can infect eggplant, lettuce, tomatoand other crops, and has a wide range of hosts (Timilsina et al. 2017; Ullah et al. 2015). To our knowledge, this is the first report of P. cichorii causing leaf spot on tobacco in China.

Characterization of Virulence Function of Pseudomonas cichorii Avirulence Protein E1 (AvrE1) during Host Plant Infection

<i>Pseudomonas cichorii</i> secretes effectors that suppress defense mechanisms in host plants. However, the function of these effectors, including avirulence protein E1 (AvrE1), in the pathogenicity of <i>P. cichorii</i>, remains unexplored. In this study, to investigate the function of <i>avrE1</i> in <i>P. cichorii</i> JBC1 (PcJBC1), we created an <i>avrE1</i>-deficient mutant (JBC1^ΔavrE1) using CRISPR/Cas9. The disease severity caused by JBC1^ΔavrE1 in tomato plants significantly decreased by reducing water soaking during early infection stage, as evidenced by the electrolyte leakage in infected leaves. The disease symptoms caused by JBC1^ΔavrE1 in the cabbage midrib were light-brown spots compared to the dark-colored ones caused by PcJBC1, which indicates the role of AvrE1 in cell lysis. The <i>avrE1</i>-deficient mutant failed to elicit cell death in non-host tobacco plants. Disease severity and cell death caused by JBC1^ΔavrE1 in host and non-host plants were restored through heterologous complementation with <i>avrE1</i> from <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 (PstDC3000). Overall, our results indicate that <i>avrE1</i> contributes to cell death during early infection, which consequently increases disease development in host plants. The roles of PcJBC1 AvrE1 in host cells remain to be elucidated.

Pseudomonas capsici sp. nov., a plant-pathogenic bacterium isolated from pepper leaf in Georgia, USA

Three phytopathogenic bacterial strains (Pc19-1T, Pc19-2 and Pc19-3) were isolated from seedlings displaying water-soaked, dark brown-to-black, necrotic lesions on pepper (Capsicum annuum) leaves in Georgia, USA. Upon isolation on King’s medium B, light cream-coloured colonies were observed and a diffusible fluorescent pigment was visible under ultraviolet light. Analysis of their 16S rRNA gene sequences showed that they belonged to the genus Pseudomonas , with the highest similarity to Pseudomonas cichorii ATCC 10857T (99.7 %). The fatty acid analysis revealed that the majority of the fatty acids were summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), C16 : 0 and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). Phylogenomic analyses based on whole genome sequences demonstrated that the pepper strains belonged to the Pseudomonas syringae complex with P. cichorii as their closest neighbour, and formed a separate monophyletic clade from other species. Between the pepper strains and P. cichorii , the average nucleotide identity values were 91.3 %. Furthermore, the digital DNA–DNA hybridization values of the pepper strains when compared to their closest relatives, including P. cichorii , were 45.2 % or less. In addition, biochemical and physiological features were examined in this study and the results indicate that the pepper strains represent a novel Pseudomonas species. Therefore, we propose a new species Pseudomonas capsici sp. nov., with Pc19-1T (=CFBP 8884T=LMG 32209T) as the type strain. The DNA G+C content of the strain Pc19-1T is 58.4 mol%.

First Report of Pseudomonas cichorii causing Bacterial Leaf Spot on Romaine lettuce (Lactuca sativa var. longifolia) and escarole (Cichorium endivia) in New Jersey.

We previously reported an outbreak of bacterial leaf spot (BLS) caused by Pseudomonas cichorii occurring on sweet basil in New Jersey during the summer of 2018 (Patel et al. 2019), a growing season characterized by increased leaf wetness due to high humidity and unusually high levels of summer rains. Leaf spot was also observed, as a one-time event, on older mature leaves on romaine and escarole lettuce during that same year. Symptoms on escarole were observed as grayish brown-to-black concentric lesions on leaf parenchyma tissue, ranging from 1 mm to 1-2 cm in diameter. In more severely diseased samples, lesions coalesced to form larger necrotic areas giving a blight appearance. On occasion, infection was observed in leaf midveins as brownish gray necrosis. Symptoms on romaine lettuce were observed mostly as coalesced blackened lesions on leaf parenchyma tissue near margins with a rotted consistency that spread to the midvein in severe cases. Margins of leaf lesions were excised and macerated in sterile water before streaking onto Nutrient Agar (NA) and King’s medium B agar (KMB) (Schaad et al. 2001). Growth on both NA and KMB were predominantly cream-colored circular bacterial colonies with undulated margins. Colonies on KMB fluoresced blue under 365nm UV light. Two representative colonies isolated from each host were selected for further characterization. All isolated tested negative for levan production, positive for oxidase, negative for potato rot, negative for arginine dihydrolase, and induced a strong hypersensitive response on tobacco within 24 h, consistent with LOPAT descriptions for P. cichorii (Lelliott et al. 1966). A single strain from each host (ESC6F2 and Rom1-1) was further characterized genetically to confirm species. PCR analysis using two primers sets: 16S rRNA gene universal primers 27F/1492R and Hcr1 primers used to amplify a 520 bp region of the pathogenicity gene cluster hrcRST in P. cichorii (Patel et al. 2019; Cottyn et al. 2011). Partial 16S rDNA gene sequences were deposited in the GenBank database for each isolate (ESC6F2: MT974180; and Rom1-1: MT982172). Sequence comparisons of ESC6F2 and Rom1-1 shared 99% identity with each other and several P. cichorii strains within the GenBank database, including strain B5-2-1 isolated from sweet basil in NJ the same year (MK501753). The partial hrcRST locus (strain ESC6F2: MW048775 and Rom1-1: MW048774) shared 100% identity to each other and strain B5-2-1 (MK507764), and 99% identity with P. cichorii strain P18-1 (MH396007) isolated from Ocimum basilicum in Hawaii. Koch’s postulate was performed on escarole var. Full Heart and Romaine lettuce var. Ideal Cos to confirm pathogenicity of the isolated strains. Bacterial suspensions (1x107 cfu/ml) were syringe-injected (0.1 ml) into the leaf midribs, and pressure infiltrated into leaf parenchyma tissue of 3 plants. Control plants were inoculated with sterile water. Blackened necrosis developed within 72 h around bacteria-inoculation points, which expanded beyond inoculation points within a week. In contrast, control plants remained healthy and symptomless. Although significant crop loss occurred due to BLS on escarole and romaine lettuce, P. cichorii has not been isolated from diseased plant material since 2018. This suggests inoculum sources did not persist beyond 2018, or favorable environmental conditions for disease are inconsistent to cause noticeable damage to New Jersey crops. References: Cottyn, B., et al. 2011. Plant Pathol. 60:453. Lelliott, R. A., et al. 1966. J. Appl. Bacteriol. 29:470. Patel, N., et al. 2019. Plant Disease. 103:2666

Identification of Pseudomonas cichorii (Swingle 1925) Stapp 1928 in hydroponic culture of lettuce in Russia

Relevance. Lettuce (Latin: Lactúca satíva) is a species of annual herbaceous plant in the genus Lettuce of the Asteraceae family. As a vegetable crop, it is cultivated everywhere in the world, and its hydroponic cultivation technology has received special development in recent years. One of the common pathogens of lettuce is Pseudomonas cichorii, causing bacterial diseases of several important cultivated plants. In this regard, the study of the occurrence of this pathogen is important.Material and methodology. The study was conducted on the basis of the Department of Agrobiotechnology of the ATI of RUDN University. The samples were provided by a commercial manufacturer of lettuce grown on a flow-through hydroponic line under conditions of minimal microbial contamination. The study of phytopathogenic bacteria includes a number of stages: isolation of bacteria on semi-selective culture media and obtaining a pure culture of bacteria; setting a test for pathogenicity (virulence); studying the phenotypic properties of bacteria; determining the taxonomic position of the isolated strains by molecular methods. All studies were conducted in accordance with the standard methods of identification of phytopathogenic bacteria.Results. As a result of the work, the distribution of the species Pseudomonas cichorii in the hydroponic culture of lettuce in the Russian Federation was confirmed. Although, according to the EPPO database, P. cichorii was first described in Russia in 1965 by microbiological methods, but isolated bacteria are not available in microbiological collections to confirm this conclusion with appropriate diagnostic methods. Twelve isolates of P. cichoriiwere studied by a biochemical and phytopathological tests, and four isolates (01, 04, 06, and 12) that showed the greatest aggressiveness on host plants and tobacco leaves were identified by DNA sequencing of the 16S rRNA gene fragment. The obtained DNA fragments showed a high similarity (99-100%) with the sequences of P. cichoriifrom the Genebank. Evaluation of the virulence of the isolated isolates on a number of other cultivated plants, and the uniformity of their biochemical characteristics showed that they represent a group of bacteria specialized in lettuce.

Pseudomonas cichorii (bacterial blight of endive).

Under environmental conditions favourable to the pathogen, diseases incited by P. cichorii cause severe damage to the host and can result in outbreaks. Outbreaks in the nursery or in the field during warm winters in Florida, USA, can lead to widespread disease affecting thousands of plants (Jones et al., 1983; Chase and Brunk, 1984; Uddin and McCarter, 1996). Diseases caused by P. cichorii can appear sporadically over a number of years then cause severe outbreaks such as the outbreak of 'varnish spot of lettuce' in California (Grogan et al. 1977) and 'brown stem of celery' in Florida, USA (Pernezny et al., 1994). Severe disease outbreaks on lettuce leading to losses of up to 100% have been reported in California (Grogan et al., 1977), Italy (Bazzi and Mazzucchi, 1979) and Portugal (Ferreira-Pinto and Oliveira, 1993). In Italy and France, P. cichorii is a recurring problem on endive and lettuce (Allex and Rat 1990; D'Ascenzo et al., 1997). In Florida, brown stem of chrysanthemum only occurs about every 5 to 6 years whereas leaf spot occurs annually (Pernezny et al., 1994).

Essential Oils from Two Apiaceae Species as Potential Agents in Organic Crops Protection

Chemical composition and herbicidal, antifungal, antibacterial and molluscicidal activities of essential oils from Choukzerk, Eryngium triquetrum, and Alexander, Smyrnium olusatrum, from western Algeria were characterized. Capillary GC-FID and GC/MS were used to investigate chemical composition of both essential oils, and the antifungal, antibacterial, molluscicidal and herbicidal activities were determined by % inhibition. Collective essential oil of E. triquetrum was dominated by falcarinol (74.8%) and octane (5.6%). The collective essential oil of S. olusatrum was dominated by furanoeremophilone (31.5%), furanodiene+curzurene (19.3%) and (E)-β-caryophyllene (11%). The E. triquetrum oil was tested and a pure falcarinol (99%) showed virtuous herbicidal and antibacterial activities against potato blackleg disease, Pectobacteriumatrosepticum, and Gram-negative soil bacterium, Pseudomonas cichorii (85 and 100% inhibition, respectively), and high ecotoxic activity against brine shrimp, Artemia salina, and the freshwater snail, Biomphalaria glabrata, with an IC50 of 0.35 µg/mL and 0.61 µg/mL, respectively. Essential oil of S. olusatrum showed interesting antibacterial and ecotoxic activity and good herbicidal activity against watercress seeds, Lepidium sativum (74% inhibition of photosynthesis, 80% mortality on growth test on model watercress), while the furanoeremophilone isolated from the oil (99% pure) showed moderate herbicidal activity. Both oils showed excellent antifungal activity against Fusarium. Both oils and especially falcarinol demonstrated good potential as new biocontrol agents in organic crop protection.

Pseudomonas cichorii (bacterial blight of endive).

Under environmental conditions favourable to the pathogen, diseases incited by P. cichorii cause severe damage to the host and can result in outbreaks. Outbreaks in the nursery or in the field during warm winters in Florida, USA, can lead to widespread disease affecting thousands of plants (Jones et al., 1983; Chase and Brunk, 1984; Uddin and McCarter, 1996). Diseases caused by P. cichorii can appear sporadically over a number of years then cause severe outbreaks such as the outbreak of 'varnish spot of lettuce' in California (Grogan et al. 1977) and 'brown stem of celery' in Florida, USA (Pernezny et al., 1994). Severe disease outbreaks on lettuce leading to losses of up to 100% have been reported in California (Grogan et al., 1977), Italy (Bazzi and Mazzucchi, 1979) and Portugal (Ferreira-Pinto and Oliveira, 1993). In Italy and France, P. cichorii is a recurring problem on endive and lettuce (Allex and Rat 1990; D'Ascenzo et al., 1997). In Florida, brown stem of chrysanthemum only occurs about every 5 to 6 years whereas leaf spot occurs annually (Pernezny et al., 1994).

D-tartrate utilization correlates with phylogenetic subclade in Pseudomonas cichorii

ABSTRACT Pseudomonas cichorii is divided into two subclades based on the 16S ribosomal DNA sequence and core genome multilocus sequence typing. It was shown that subclade 2 strains utilize D-tartrate as a sole carbon source, whereas subclade 1 strains do not. Draft genome sequencing was performed with P. cichorii strains to identify D-tartrate utilization genes. By genome comparative and homology search studies, an approximately 7.1 kb region was identified to be involved in D-tartrate utilization. The region is subclade 2 specific, and contains tarD and dctA genes, which encode a putative enzyme and transporter of D-tartrate, respectively. When the region was introduced into subclade 1 strains, the transformants were able to utilize D-tartrate. Partial fragments of tarD and dctA were amplified from all subclade 2 strains tested in this study by PCR using gene specific primers, but not from subclade 1 strains. This is the first report on the genetic analysis of biochemical characteristics corresponding to a specific phylogenetic group in P. cichorii.