Seed microbiota revealed by a large-scale meta-analysis including 50 plant species

Seed microbiota constitutes a primary inoculum for plants that is gaining attention due to its role for plant health and productivity. Here, we performed a meta-analysis on 63 seed microbiota studies covering 50 plant species to synthesize knowledge on the diversity of this habitat. Seed microbiota are diverse and extremely variable, with taxa richness varying from one to thousands of taxa. Hence, seed microbiota presents a variable (i.e flexible) microbial fraction but we also identified a stable (i.e. core) fraction across samples. Around 30 bacterial and fungal taxa are present in most plant species and in samples from all over the world. Core taxa, such as Pantoea agglomerans, Pseudomonas viridiflava, P. fluorescens, Cladosporium perangustum and Alternaria sp., are dominant seed taxa. The characterization of the core and flexible seed microbiota provided here will help uncover seed microbiota roles for plant health and design effective microbiome engineering.

Within-Plant Variation in Rosmarinus officinalis L. Terpenes and Phenols and Their Antimicrobial Activity against the Rosemary Phytopathogens Alternaria alternata and Pseudomonas viridiflava

This study investigated within-plant variability of the main bioactive compounds in rosemary (Rosmarinus officinalis L.). Volatile terpenes, including the enantiomeric distribution of monoterpenes, and phenols were analyzed in young and mature foliar, cortical and xylem tissues. In addition, antimicrobial activity of rosmarinic acid and selected terpenes was evaluated against two rosemary pathogens, Alternaria alternata and Pseudomonas viridiflava. Data showed that total concentration and relative contents of terpenes changed in relation to tissue source and age. Their highest total concentration was observed in the young leaves, followed by mature leaves, cortical and xylem tissues. Rosmarinic acid and carnosic acid contents did not show significant differences between leaf tissues of different ages, while young and mature samples showed variations in the content of four flavonoids. These results are useful for a more targeted harvesting of rosemary plants, in order to produce high-quality essential oils and phenolic extracts. Microbial tests showed that several terpenes and rosmarinic acid significantly inhibited the growth of typical rosemary pathogens. Overall, results on antimicrobial activity suggest the potential application of these natural compounds as biochemical markers in breeding programs aimed to select new chemotypes less susceptible to pathogen attacks, and as eco-friendly chemical alternatives to synthetic pesticides.

First Report of Leaf Blight of Adenophora triphylla var. japonica caused by Pseudomonas viridiflava in Korea

Severe disease with leaf spots and necrotic symptoms were observed in Adenophora triphylla var. japonica (Regel) Hara (A. triphylla) during the survey in July 2020 on a field in Andong, Gyeongbuk province, Korea. It is a highly valued medicinal plant used to treat various diseases, including cough, cancer, and obesity. The infected plants initially showed spots with halo lesions, at later stages, enlarged and spread to the leaves, which the lesions becoming yellowing and chlorotic (Fig. 1). In some areas, disease incidence was up to 15% of the plants. The symptomatic samples were collected from A. triphylla and cut into 4 to 5 mm squares, surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed three times, and macerated in sterile distilled water (SDW). They were spread onto nutrient agar (NA) plates and incubated at 28°C for 3 days. The representative bacterial strains selected for identification showed fluorescent colonies on King’s medium B (KB). Fifteen isolates from independent samples were subjected to biochemical and pathogenicity tests. The isolates induced a hypersensitive reaction in tobacco leaves, gave a reaction in the anaerobe respiratory test, and were negative for levan, oxidase, arginine dihydrolase, gelatin hydrolysis, aesculin hydrolysis, and starch hydrolysis. The isolated strains presented the following LOPAT profile: – – + – +. The Biolog GN2 microplate and the Release 4.20 system putatively found the isolate to exhibit 93% similarity with the bacterium, Pseudomonas viridiflava. Likewise, analysis of FAME profiles using the Microbial identification system (Sherlock version 3.1) also characterized the representative bacterial strain as P. viridiflava with 87% similarity. The genomic DNA of the isolate was extracted, and the 16S rDNA sequence was amplified with a universal bacterial primer set (27F and 1492R). The sequence was submitted to GenBank under the accession number MT975233. BLASTn analysis yielded 99.79% identity with P. viridiflava strain RT228.1b (accession no. AY604846.1) and 99.72% similarity with P. viridiflava KNOX249.1b strain (accession no. AY604848.1). Phylogenetic dendrogram constructed from the comparative analysis of 16S rDNA gene sequences showing the relationship between P. viridiflava GYUN274 and related Pseudomonas species (Fig. 2). Pathogenicity tests were conducted three times on seedling of A. triphylla by spraying 50 ml of bacterial suspensions of a 24-h culture in KB medium (108 CFU/ml). The leaves inoculated with SDW alone did not develop symptoms; however, the plants treated with isolated bacterial suspensions developed halo and blight symptoms similar to those observed in the field 7 days post-inoculation. Finally, Koch’s postulates were verified by re-isolating P. viridiflava from all symptomatic tissues and determined to be morphologically identical to the original isolates. To our knowledge, this is the first report of leaf blight disease of A. triphylla caused by P. viridiflava in Korea. Based on the observed symptoms, and identification by morphological characteristics, molecular data, and pathogenicity against the host plant, the proper control measures can be identified in future studies.

Pseudomonas viridiflava (bacterial leaf blight of tomato).

P. viridiflava may very occasionally cause significant crop damage, though it is commonly isolated as a sub-population in the investigation of more vigorous pathogens.

First report of bacterial leaf spot disease on sunflower (Helianthus annuus) caused by Pseudomonas viridiflava in South Korea

In September 2019, bacterial leaf spot symptoms were observed on sunflowers in an experimental field in Eumseong, South Korea. The leaves of infected plants initially showed irregular brown spots surrounded by haloes; as the disease progressed, the spots became enlarged and darkened (eXtra Fig. 1a). At the flowering stage, leaves became dry and showed signs of blight including defoliation; dark brown spots were also observed on sunflower stems and petioles but not on floral discs (eXtra Fig. 1b). Disease incidence ranged from 5% to 30% in three surveyed plots of the field. Symptomatic leaf tissue was surface-sterilized, macerated with sterile distilled water, and cultured on nutrient agar plates at 28°C for 48 h. After incubation, nine bacterial isolates, representing individually collected samples from each field, were selected for further study. All nine isolates were Gram-negative and fluorescent pigments produced under UV on King’s medium B. With the LOPAT test, the isolates were levan negative, oxidase negative, positive for pectinolytic activity, arginine dihydrolase negative, and positive for tobacco hypersensitivity. Based on 16s rRNA sequences, all isolates shared 100% identity with Pseudomonas viridiflava strain KNOX209.1 (GenBank accession no. AY604847). The 16s rRNA sequences of nine isolates were deposited in GeneBank (accession nos. MT393747, MW446479 to MW446486). Based on the phylogenetic analysis of the 16s rRNA, all isolates were grouped with P. viridiflava strains isolated from globe artichoke, Chinese cabbage, and rape (Myung et al. 2010, Sanver et al. 2019, and Liu et al. 2019). The isolates CPB 19362, CPB 19366 and CPB 19372, which represent each plot were selected for further phylogenetic analysis and pathogenicity assays. The identity of these isolates was confirmed by sequences of housekeeping genes of the gyrase B subunit (gyrB) and RNA polymerase σ70 factor (rpoD) (Yamamoto et al. 2000) (GenBank accession nos. MT409400, MW446487 and MW446494 for gyrB and MT409401, MW446495 and MW446502 for rpoD). Based on the phylogenetic analyses of gyrB and rpoD, the three isolates belong to the same clade as the P. viridiflava pathotypes and were distinguished from P. syringae complex (eXtra Fig. 2). These results indicated that the bacteria isolated from the spots on the sunflower plants were P. viridiflava strains. To confirm the pathogenicity, bacterial suspensions (approximately 108 CFU/mL) of three representative isolates sprayed onto 4-week-old sunflower (cv. Common) seedlings separately until runoff occurred. Sterile distilled water was used as a control and inoculated in the same manner. After inoculation, plants were covered with transparent plastic bags at room temperature for 24 h. Plastic bags were then removed and plants were grown on a plant growth shelf at 25°C in 50% relative humidity. The leaves of plants inoculated with the bacterial suspensions developed small brown spots after 24 h. After 3 days, brown spots surrounded by chlorotic or necrotic areas were observed on infected leaves (eXtra Fig. 1c). These spots gradually increased in size and formed brown lesions with haloes similar to those of infected field-grown plants (eXtra Fig. 1d), but not on the controls treated with sterile water. The pathogenicity test was repeated three times. Isolates recovered from infected leaves showed the same morphological, biochemical, and molecular characteristics as the original isolates from field samples. To our knowledge, this is the first report of bacterial leaf spot on sunflower caused by P. viridiflava in South Korea.

The first report of bacterial streak and rot of onion caused by Pseudomonas viridiflava in Japan.

In March 2020, a bacterial streak and rot symptom was observed on the onion (Allium cepa L.) leaves in Akita Prefecture of Japan. On the beginning, oval and dark-greenish water-soaked lesions with grayish-white necrotic center, 2-3 mm in diameter, appeared on the middle or the tip of upper leaves. Lesions, frequently surrounded by light yellow halo, expanded along veins and overlapped together. As lesions grew, the center of the lesions turned to light brown necrosis. The basal areas of diseased leaves often rotted, causing the withering of a whole leaf at last. From the water-soaked tissues of young lesions, a bacterium forming cream white colonies and producing fluorescent pigment on King’s medium B was consistently isolated, and suggested to be a member of genus Pseudomonas. The isolates were positive for potato soft rot and tobacco hypersensitive reaction, and negative for levan production, oxidase and arginine dihydrolase activity, indicating that they belonged to LOPAT group II, Pseudomonas viridiflava, defined by Lelliott et al. (1966). P. viridiflava is known as a pathogen of bacterial streak and bulb rot of onion in United States and Uruguay, though it has not been reported in Japan. Four isolates were chosen for further examinations. Pathogenicity tests on onion leaves stubbed with selected isolates reproduced similar symptoms as observed in field's samples. After 2 days of inoculation, isolates produced water-soaking around the stabbed holes, developed into grayish-white necrosis on 4 days after inoculation. After 10 days of inoculation, lesions grew to necrotic streaks with light blown center surrounded by water-soaking and light yellow halo. Additionally, on onion scales, isolates caused water-soaked rot, yellow- to brown-colored, in 1-2 days after inoculation. From water-soaked areas of both of leaves and scales, inoculum was reisolated and fulfilled Koch´s postulates. In phenotypic properties, isolates showed consistent results as P. viridiflava strains identified in former studies. They were gram negative and aerobic, positive for hydrolysis of esculin, casein and gelatin, and negative for nitrate reduction and indole production. Regarding the utilization of carbohydrates, positive for 2-keto-gluconate, glucose, fructose, D-galactose, mannose, L(+)arabinose, glycerol, mannitol, sorbitol, myo-inositol, meso-erythritol, meso-tartrate, D(-)-tartrate, gluconate, n-caprate, dl-malate, citrate, L-arginine, L-aspartate, L-glutamate, and negative for D-arabinose, maltose, sucrose, raffinose, adonitol, trehalose, L-tartrate, L-rhamnose, acetate. The 16S rRNA gene sequences (Frank et al 2008) of four isolates (1,377 bp) showed 100% similarity as that of P. viridiflava type strain LMG 2352T (GenBank Accession No. Z76671) in BLASTN search. In phylogenetic analysis using gyrB (910 bp) and rpoD (801 bp) genes (Maeda et al 2006), isolates formed a cluster with P. viridiflava strains deposited in public databases, independent from other closely related Pseudomonas species. Sequences of 16S rRNA (GenBank Accession Nos. LC597475-LC597478), gyrB (LC597479-LC597482) and rpoD (LC597483-LC597486) genes were deposited in DNA Data Bank of Japan. According to these results, the isolates were identified as P. viridiflava (Burkholder 1930) Dowson 1939. This is the first report of the occurrence of bacterial streak and rot of onion caused by P. viridiflava in Japan, causing severe damage on onion growth.