Effects of ‘Candidatus Phytoplasma asteris’ on the Volatile Chemical Content and Composition of Grindelia robusta Nutt.

Grindelia robusta, a perennial herb, contains an essential oil that is used as an antitussive, sedative, and analgesic agent. During the spring of 2007, ‘Candidatus Phytoplasma asteris’‐related phytoplasmas were identified in plants showing virescence and phyllody symptoms. The qualitative and quantitative composition of the oil of healthy and infected plants was compared by gas chromatography/mass spectrometry. Samples from six symptomatic and five asymptomatic plants tested by nested PCR followed by RFLP analyses confirmed the presence of ‘Ca. P. asteris’ in all symptomatic samples. The oils from healthy and infected plants, obtained by steam distillation, contained 42 components; that of healthy plants contained a higher concentration of monoterpenes, especially limonene and bornyl acetate, which were nearly 50% higher.     

Biological and molecular evidence for the transmission of aster yellows phytoplasma to French marigold (Tagetes patula) by the flatid planthopper Metcalfa pruinosa

Aster yellows (AY) phytoplasmas (Candidatus Phytoplasma asteris) are associated with a number of plant diseases throughout the world. Several insect vectors are responsible for spreading AY diseases resulting in wide distribution and low host specificity. Because the role of sucking insects as vectors of phytoplasmas is widely documented, and the citrus flatid planthopper Metcalfa pruinosa is a phloem feeder, it has been incriminated as a possible vector of phytoplasmas. However, its ability to transfer phytoplasma has not been confirmed. The present work shows that M. pruinosa (Hemiptera: Flatidae), a polyphagous planthopper, is able to vector Ca. P. asteris to French marigold (Tagetes patula). Transmission experiments were conducted in 2017 and 2018 in central Hungary by two approaches: (a) AY-infected M. pruinosa were collected from an area with severe incidence of the disease on T. patula and caged on test plants for an inoculation-access period of 2 weeks, and (b) presumably phytoplasma-free insects were collected from apparently healthy grapevines (Vitis vinifera L.) and fed on AY-infected T. patula plants for 2 weeks prior to being caged on test plants. AY disease symptoms developed on 4 out of 10 and 10 out of 15 test plants, respectively. All phytoplasma-positive marigold and M. pruinosa samples showed identical RFLP patterns and shared 100% 16S rDNA sequence identity with each other and with the aster yellows phytoplasma strain AJ33 (GenBank accession number MK992774). These results indicated that the phytoplasma belonged to the phytoplasma subgroup 16SrI-B Ca. P. asteris. Therefore, the work presented here provides experimental evidence that M. pruinosa is a vector of a 16SrI-B subgroup phytoplasma to T. patula.     

Biological activities of two fungistatic antibiotics produced by Bacillus cereus UW85.

Cultures and culture filtrates of Bacillus cereus UW85 suppress damping-off of alfalfa caused by Phytophthora medicaginis. We studied the role in disease suppression of two antibiotics from culture filtrates of UW85 that reversibly inhibited growth of P. medicaginis. We purified the two antibiotics by cation-exchange chromatography and high-voltage paper electrophoresis and showed that one of them, designated zwittermicin A, was an aminopolyol of 396 Da that was cationic at pH 7.0; the second, designated antibiotic B, appeared to be an aminoglycoside containing a disaccharide. Both antibiotics prevented disease of alfalfa seedlings caused by P. medicaginis. Purified zwittermicin A reversibly reduced elongation of germ tubes derived from cysts of P. medicaginis, and antibiotic B caused swelling of the germ tubes. Mutants generated with Tn917 or mitomycin C treatment were screened either for antibiotic accumulation in an agar plate diffusion assay or for the ability to suppress damping-off disease of alfalfa. Of 2,682 mutants screened for antibiotic accumulation, 5 mutants were substantially reduced in antibiotic accumulation and disease-suppressive activity. Of the 1,700 mutants screened for disease-suppressive activity, 3 mutants had reduced activity and they accumulated less of both antibiotics than did the parent strain. The amount of antibiotic accumulated by the mutants was significantly correlated with the level of disease suppression. Addition of either zwittermicin A or antibiotic B to alfalfa plants inoculated with a culture of a nonsuppressive mutant resulted in disease suppression. These results demonstrate that B. cereus UW85 produces two fungistatic antibiotics that contribute to suppression of damping-off disease of alfalfa.     

Salt resistance and synergistic effect with vancomycin of alpha-helical antimicrobial peptide P18.

P18 (KWKLFKKIPKFLHLAKKF-NH(2)) is an alpha-helical antimicrobial peptide designed from a cecropin A-magainin 2 hybrid. In this study, P18 was found to show strong antimicrobial activity against several antibiotic-resistant bacterial and fungal strains. Both the salt resistance on antimicrobial activity and the synergistic effect with clinically used antibiotic agents are critical factors in developing effective peptide antibiotic drugs. For this reason, we investigated the salt resistance of P18 to antagonism by NaCl, CaCl(2), and MgCl(2) on antimicrobial activity and the synergistic effect of P18 with vancomycin against vancomycin-resistant Enterococcus faecium (VREF). Compared to magainin 2, P18 showed strong resistance on antimicrobial activity against bacterial strains and C. albicans under high NaCl concentrations of 100-200 mM. In addition, P18 displayed much greater salt resistance on antibacterial activity against Gram-negative bacteria at the physiological or elevated concentrations of CaCl(2) and MgCl(2) than magainin 2. Furthermore, the combination study revealed that P18 has a relatively effective synergistic effect with vancomycin against VREF. Thus, these results support that P18 may prove to be a salt-resistant antibiotic peptide potentially useful in the treatment of cystic fibrosis patients as well as a valuable adjuvant for antimicrobial chemotherapy.     

Detection and identification of ‘Candidatus Phytoplasma asteris’ in Brassica rapa subsp. pekinensis in Poland

Severe growth abnormalities, including leaf yellowing, sprout proliferation and flower virescence and phyllody, were found on Brassica rapa subsp. pekinensis plants in Poland. The presence of phytoplasma in naturally infected plants was demonstrated by polymerase chain reaction assay employing phytoplasma universal P1/P7 followed by R16F2n/R16R2 primer pairs. The detected phytoplasma was identified using restriction fragment length polymorphism analysis (RFLP) of the 16S rRNA gene fragment with AluI, HhaI, MseI and RsaI endonucleases. After enzymatic digestion, all tested samples showed restriction pattern similar to that of ‘Candidatus phytoplasma asteris’. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Sequences of the 16S rDNA gene fragment of analysed phytoplasma isolates were nearly identical. They revealed high nucleotide sequence identity (>98%) with corresponding sequences of other phytoplasma isolates from subgroup 16SrI‐B, and they were classified as members of ‘Candidatus phytoplasma asteris’. This is the first report of the natural occurrence of phytoplasma‐associated disease in plants of Chinese cabbage.     

Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed.     

Lineage-specific decay of folate biosynthesis genes suggests ongoing host adaptation in phytoplasmas

Phytoplasmas are nonculturable cell wall-less, obligate intracellular pathogens of plants and insect vectors. In their descent from walled bacterial ancestors, phytoplasmas underwent massive genome reduction, resulting in some of the smallest cellular genomes known in nonsymbiotic bacteria. While requirements for in vitro culture of phytoplasmas remain unknown, two opposing reports have appeared concerning genes encoding the ability of phytoplasmas to synthesize folates de novo. One study found pseudogene homologs of folP and folK, obviating folate synthesis in "Candidatus Phytoplasma asteris"-related strain CPh, whereas, a separate study found intact genes encoding a complete folate biosynthesis pathway in "Ca. Phytoplasma asteris"-related strain OY. To resolve the apparent conflict, we hypothesized that evolutionary adaptation to the availability of folate and/or other metabolites in host cells is an ongoing process in the phytoplasma clade that is reflected in part by differences among phytoplasmas in the status of genes of the folate biosynthesis pathway. By studying folP and folK loci in 11 closely related phytoplasmas, we determined that these essential folate biosynthesis genes are intact in some phytoplasmas but are deteriorating in closely related strains. We suggest that the status of the folate biosynthesis pathway and the course of gene decay are lineage-specific, predicting the eventual, lineage-related loss of recognizable folP and folK homologs in phytoplasma genomes.     

Comparative genome analysis of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A) and "Ca. Phytoplasma asteris" Strains OY-M and AY-WB

The chromosome sequence of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of "Ca. Phytoplasma australiense" with "Ca. Phytoplasma asteris" strains OY-M and AY-WB showed that the gene order was more conserved between the closely related "Ca. Phytoplasma asteris" strains than to "Ca. Phytoplasma australiense." Differences observed between "Ca. Phytoplasma australiense" and "Ca. Phytoplasma asteris" strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function.     

Identification of four distinct ‘Candidatus Phytoplasma’ species in pomegranate trees showing witches' broom,little leaf and yellowing in Jordan,and preliminary insights on their putative insect vectors and reservoir plants

During field surveys conducted in northern Jordan from June to November 2020, phytoplasma-like symptoms, including leaf yellowing/reddening and rolling, little leaf and witches' broom were observed in pomegranate. Disease incidence in 22 surveyed orchards ranged from 30% to 65%. Nested PCR-based amplification of 16S rRNA gene detected phytoplasmas in 17% of collected symptomatic pomegranate trees. Amplicon nucleotide sequence analyses allowed attributing the detected phytoplasmas to ‘Candidatus Phytoplasma solani’, ‘Ca. P. aurantifolia’, ‘Ca. P. asteris’ and ‘Ca. P. ulmi’. These phytoplasmas were found in plants showing specific symptoms and differentially distributed in the considered locations. Additionally, three cicadellids (Macrosteles sexnotatus, Cicadulina bipunctata and Psammotettix striatus) and two non-crop plants (Plantago major and Capsicum annuum) resulted hosting ‘Ca. P. asteris’ strains, and one cicadellid (Balclutha incisa) was carrying a ‘Ca. P. solani’ strain. A new pomegranate disease complex associated with multiple phytoplasmas, including ‘Ca. P. aurantifolia’ and ‘Ca. P. ulmi’, never reported before in this host plant, is described here. Moreover, preliminary indications are provided on its possible epidemiology in Jordan, involving two putative insect vectors (M. sexnotatus, B. incisa) first reported in the Country.     

‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma aurantifolia’, New Phytoplasma Species Infecting Apple Trees in Iran

During several surveys in extensive areas in central Iran, apple trees showing phytoplasma diseases symptoms were observed. PCR tests using phytoplasma universal primer pairs P1A/P7A followed by R16F2n/R16R2 confirmed the association of phytoplasmas with symptomatic apple trees. Nested PCR using 16SrX group‐specific primer pair R16(X)F1/R1 and aster yellows group‐specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy indicated that apple phytoplasmas in these regions did not belong to the apple proliferation group, whereas aster yellows group‐related phytoplasmas caused disease on some trees. Restriction fragment length polymorphism (RFLP) analyses using four restriction enzymes (HhaI, HpaII, HaeIII and RsaI) and sequence analyses of partial 16S rRNA and rp genes demonstrated that apple phytoplasma isolates in the centre of Iran are related to ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’. This is the first report of apples infected with ‘Ca. Phytoplasma asteris’ in Iran and the first record from association of ‘Ca. Phytoplasma aurantifolia’ with apples worldwide.     

Antibacterial, antitumor and hemolytic activities of alpha-helical antibiotic peptide, P18 and its analogs.

The alpha-helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF-NH2) designed from the cecropin A(1-8)-magainin 2 (1-12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro9 residue at central position of P18 on cell selectivity was investigated by Pro9 --> Leu or Pro9 --> Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in alpha-helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure-antibiotic activity relationships of P18, a series of N- or C-terminal deletion and substitution analogs of P18 were synthesized. The C-terminal region of P18 was related to its antibiotic activity and alpha-helical conformation on lipid membranes rather than N-terminal one. Higher alpha-helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L9]-P18 and [S9]-P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N-1, N-2, N-3, N-3L and N-4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti-infective or antitumor agents.     

Biochemical and epigenetic changes in phytoplasma‐recovered periwinkle after indole‐3‐butyric acid treatment

Aim: To elucidate the possible mechanism of phytoplasma elimination from periwinkle shoots caused by indole‐3‐butyric acid (IBA) treatment. Methods and Results: It has been shown that a transfer of in vitro‐grown phytoplasma‐infected Catharanthus roseus (periwinkle) plantlets from medium supplemented with 6‐benzylaminopurine (BA) to one supplemented with IBA can induce remission of symptoms and even permanent elimination of ‘Candidatus Phytoplasma asteris’ reference strain HYDB. Endogenous auxin levels and general methylation levels in noninfected periwinkles, periwinkles infected with two ‘Candidatus Phytoplasma’ species and phytoplasma‐recovered periwinkles were measured and compared. After the transfer from cytokinin‐ to auxin‐containing media, healthy shoots maintained their phenotype, methylation levels and hormone concentrations. Phytoplasma infection caused a change in the endogenous indole‐3‐acetic acid to IBA ratio in periwinkle shoots infected with two ‘Candidatus Phytoplasma’ species, but general methylation was significantly changed only in shoots infected with ‘Ca. P. asteris’, which resulted in the only phytoplasma species eliminated from shoots after transfer to IBA‐containing medium. Both phytoplasma infection and treatment with plant growth regulators influenced callose deposition in phloem tissue, concentrations of photosynthetic pigments and soluble proteins, H₂O₂ levels and activities of catalase (CAT) and ascorbate peroxidase (APX). Conclusion: Lower level of host genome methylation in ‘Ca. P. asteris’‐infected periwinkles on medium supplemented with BA was significantly elevated after IBA treatment, while IBA treatment had no effect on cytosine methylation in periwinkles infected with ‘Candidatus Phytoplasma ulmi’ strain EY‐C. Significance and Impact of the Study: Hormone‐dependent recovery is a distinct phenomenon from natural recovery. As opposed to spontaneously recovered plants in which elevated peroxide levels and differential expression of peroxide‐related enzymes were observed, in hormone‐dependent recovery changes in global host genome, methylation coincide with the presence/absence of phytoplasma.     

The radiobiological characteristics of the P+ variant of Bacillus brevis var. G.--B. in relation to gramicidin S synthesis]

The radiosensitivity of P(+) variant Bacillus brevis var. G.-B. cells cultured under condition of normal and inhibited gramicidin S synthesis, antibiotically high-active strain and high radioresistant cells has been studied. It has been shown that the radioresistance of bacterial cells correlates, in general, with their antibiotic activity: the antibiotic superproduced is more radioresistant than P(+) variant, the inhibition of antibiotic synthesis by beta-phenil-beta-alanin rises a little the sensitivity of P(+) variant cells. But the radioresistant fraction of P(+) variant contains the lower antibiotic amount than the whole population. It has been concluded that the radioprotective action of gramicidin S can not be the only reason of the above-mentioned differences in radiosensitivity.     

First Report of Phytoplasma (16SrI) Associated with Yellow Decline Disease of Royal Palms [Roystonea regia (Kunth) O. F. Cook] in Malaysia

Royal Palms (Roystonea regia) with symptoms such as severe chlorosis, stunting, collapse of older fronds and general decline were observed in the state of Selangor, Malaysia. Using polymerase chain reaction (PCR) amplification with phytoplasma universal primer pair P1/P7 followed by R16F2N/R16R2 and fU5/rU3 as nested PCR primer pairs, all symptomatic plants tested positively for phytoplasma. Results of phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences revealed that the phytoplasma associated with Royal Palm yellow decline (RYD) was an isolate of ‘Candidatus Phytoplasma asteris’ belonging to a new 16SrI‐subgroup. These results show that Roystonea regia is a new host for the aster yellows phytoplasma (16SrI). This is the first report on the presence of 16SrI phytoplasma on Royal Palm trees in Malaysia.     

Metabolic activity of Staphylococcus epidermidis is high during initial and low during late experimental foreign-body infection

Foreign-body infection (FBI) is notoriously resistant to eradication by antibiotic treatment. It is hypothesized that reduced bacterial metabolic activity contributes to this resistance. We examined the metabolic activity of Staphylococcus epidermidis in 204 samples recovered during in vitro foreign-body colonization and in 424 samples recovered during in vivo FBI in a rat model. Metabolic activity was measured by determining the amount of 16S rRNA per genome by quantitative PCR. The initial foreign-body-associated growth proved to be a metabolically active process, both in vitro and in vivo. The initial 16S rRNA content was similar to that observed during in vitro exponential-growth phase. However, during late in vivo FBI, a 114-fold (P < 0.0001) decrease in the 16S rRNA content was observed, indicating that there was markedly decreased metabolic activity. This decreased metabolic activity during late FBI can explain at least in part why such infections are so difficult to eradicate with conventional antibiotic treatment.     

Effect of ultraviolet-induced mutants of Trichoderma harzianum with altered antibiotic production on selected pathogens in vitro

Mutants of Trichoderma harzianum with altered antibiotic production were isolated using ultraviolet light mutagenesis. These included strains whose activity in a Fusarium oxysporum spore germination assay was greater than twice that of the parental strain and one that had no detectable antifungal activity. Characterisation of extracellular metabolites of these strains using thin-layer chromatography and gas-liquid chromatography showed that the strains with high activity produced only elevated levels of a 6-n-pentyl pyrone, the antibiotic produced by the parental strain, but two new antifungal compounds. One of these has been identified as an isonitrile antibiotic. The nature of the interactions of the mutants with Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum was examined in an in vitro dual-plating assay using two media. High antibiotic production by two T. harzianum strains, BC10 and BC63, did increase inhibition of hyphal growth of R. solani and P. ultimum, but there was no correlation between increased antibiotic production and colonisation ability. In some cases the increased antibiotic levels appeared to impede colonisation of F. oxysporum and R. solani by the mutants. Slow growth rate also affected colonising ability. The types of interactions showed great variability depending on the nature of the T. harzianum isolate and on the test fungus.     

Biological properties of a Bacillus brevis 101 mutant

Some features of the Bacillus brevis 101 mutant producing the antibiotic gramicidin S are described. The mutant is very close to the initial P+-variant of Bacillus brevis var. G-B by cultural, physiological and biochemical characteristics. The most typical features of Bacillus brevis 101 are high antibiotic activity (up to 2 g/l) and the specific phenotype of the colonies. The phenotypical features of Bacillus brevis 101 are dependent on the conditions of its cultivation. On minimal media rich in organics a change of the culture correlated with a lower antibiotic activity.     

Biosynthesis of amphotericin derivatives lacking exocyclic carboxyl groups

Amphotericin B is a medically important antifungal antibiotic that is also active against human immunodeficiency virus, Leishmania parasites, and prion diseases. The therapeutic use of amphotericin B is restricted by severe side effects that can be moderated by liposomal formulation or structural alteration. Chemical modification has shown that suppression of charge on the exocyclic carboxyl group of amphotericin B substantially reduces toxicity. We report targeted deletions of the amphN cytochrome P450 gene from the chromosome of the amphotericin-producing bacterium Streptomyces nodosus. The mutant strains produced amphotericin analogues in which methyl groups replace the exocyclic carboxyl groups. These compounds retained antifungal activity and had reduced hemolytic activity.