Suppression of Root Rot Fungal Diseases in Common Beans (Phaseolus vulgaris L.) through the Application of Biologically Synthesized Silver Nanoparticles.

The biosynthesis of silver nanoparticles (AgNPs) using plant extracts has become a safe replacement for conventional chemical synthesis methods to fight plant pathogens. In this study, the antifungal activity of biosynthesized AgNPs was evaluated both in vitro and under greenhouse conditions against root rot fungi of common beans (Phaseolus vulgaris L.), including Macrophomina phaseolina, Pythium graminicola, Rhizoctonia solani, and Sclerotium rolfsii. Among the eleven biosynthesized AgNPs, those synthesized using Alhagi graecorum plant extract displayed the highest efficacy in suppressing those fungi. The findings showed that using AgNPs made with A. graecorum at a concentration of 100 µg/mL greatly slowed down the growth of mycelium for R. solani, P. graminicola, S. rolfsii, and M. phaseolina by 92.60%, 94.44%, 75.93%, and 79.63%, respectively. Additionally, the minimum inhibitory concentration (75 µg/mL) of AgNPs synthesized by A. graecorum was very effective against all of these fungi, lowering the pre-emergence damping-off, post-emergence damping-off, and disease percent and severity in vitro and greenhouse conditions. Additionally, the treatment with AgNPs led to increased root length, shoot length, fresh weight, dry weight, and vigor index of bean seedlings compared to the control group. The synthesis of nanoparticles using A. graecorum was confirmed using various physicochemical techniques, including UV spectroscopy, Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS) analysis. Collectively, the findings of this study highlight the potential of AgNPs as an effective and environmentally sustainable approach for controlling root rot fungi in beans.

Systemic Resistance Induction of Potato and Tobacco Plants against Potato Virus Y by Klebsiella oxytoca.

Potato Virus Y (PVY) is a serious potato disease that may significantly decrease potato production. To suppress potato virus infection, several measures have been undertaken. The utilization of plant growth-promoting rhizobacteria is one of these methods. Biochar soil treatment is believed to provide plants with a number of advantages, including increased plant growth and the development of systemic resistance to a variety of plant diseases. The goal of this research was to see whether adding biochar and Klebsiella oxytoca to the soil might cause PVY resistance and enhance the involved mechanisms in PVY resistance. Potato and tobacco seedlings treated with Klebsiella oxytoca and biochar exhibited the same impact of significant symptom reduction, with complete negative ELISA findings, supporting the antiviral activity of K. oxytoca and biochar. Furthermore, owing to the connection between the ISR implicated substrates, significant amounts of polyphenol oxidase, catalase, and superoxide dismutase were observed in treated plants, with the same behavior as defense genes expression levels. It may be a step forward in the development of biochar and K. oxytoca as potential environmentally friendly disease control strategies against PVY.

Resistance Induction and Direct Antifungal Activity of Some Monoterpenes against Rhizoctonia solani, the Causal of Root Rot in Common Bean.

This study was conducted to evaluate eco-friendly control agents (carvone, cuminaldehyde, and linalool) against Rhizoctonia solani, which causes root rot disease either by induction of defense response or direct antifungal activity. The induction of resistance was examined by detecting the transcription of defense genes and the effect of the tested control agents on the growth and the yield of common bean plants. The growth of R. solani was significantly inhibited after treatment with the tested compounds compared to the untreated control under laboratory conditions. The disease severity of root rot was decreased in common bean plants treated with the tested compounds compared to untreated control plants under greenhouse conditions. Common bean plants treated with the tested control agents expressed defense genes (Phenylalanine ammonia lyase and ß-1,3-Glucanase) involved in jasmonic acid (JA) and salicylic acid (SA) signaling pathways with 2-5 fold higher than the control. Treatment of common beans with the tested control agents and fungicide significantly improved the growth and yield characteristics of common bean. Therefore, the use of monoterpenes could be a novel strategy to control this pathogen and consider the first report.

Induction of Systemic Resistance against Sheath Blight in Rice by Different Pseudomonas Isolates.

Sheath blight disease is a fungal pathogen that causes leaf blight in rice plants, resulting in significant yield losses throughout the growing season. Pseudomonas spp. have long been used as biocontrol agents for a variety of plant diseases. Four Pseudomonas isolates were tested for their ability to promote rice growth and generate systemic resistance to Rhizoctonia solani, the causal pathogen of sheath blight disease. In vitro, Pseudomonas isolates produced the growth hormone indole acetic acid (0.82-1.82 mg L-1). Additionally, seed treatment with Pseudomonas putida suspension outperformed P. brassicacearum, P. aeruginosa and P. resinovorans in terms of germination and vigor evaluation. The maximum seed germination of 89% was recorded after seed treatments with a fresh suspension of P. putida, followed by 87% germination in P. aeruginosa treatment, compared with only 74% germination in the untreated controls. When compared with the infected control plants, all Pseudomonas isolates were non-pathogenic to rice and their co-inoculation considerably enhanced plant growth and health by reducing the disease index to 37% and improving plant height (26%), fresh weight (140%) and dry weight (100%). All Pseudomonas isolates effectively reduced sheath blight disease incidence, as well as the fungicide carbendazim, which is recommended for field management of R. solani. In comparison to untreated control seedlings, treatment with Pseudomonas isolates enhanced the production of peroxidase and polyphenol oxidase enzymes and the expression of the phenylalanine ammonia lyase (PAL) and NPR1 genes, which could be involved in disease incidence reduction. In conclusion, the use of Pseudomonas spp. has been demonstrated to improve rice growth and resistance to R. solani while also providing an environmentally acceptable option to the agroecosystems.

Mechanism of Wheat Leaf Rust Control Using Chitosan Nanoparticles and Salicylic Acid.

Wheat leaf rust is one of the world's most widespread rusts. The progress of the disease was monitored using two treatments: chitosan nanoparticles and salicylic acid (SA), as well as three application methods; spraying before or after the inoculation by 24 h, and spraying both before and after the inoculation by 24 h. Urediniospore germination was significantly different between the two treatments. Wheat plants tested for latent and incubation periods, pustule size and receptivity and infection type showed significantly reduced leaf rust when compared to untreated plants. Pucciniatriticina urediniospores showed abnormalities, collapse, lysis, and shrinkage as a result of chitosan nanoparticles treatment. The enzymes, peroxidase and catalase, were increased in the activities. In both treatments, superoxide (O2-) and hydrogen peroxide (H2O2), were apparent as purple and brown discolorations. Chitosan nanoparticles and SA treatments resulted in much more discoloration and quantitative measurements than untreated plants. In anatomical examinations, chitosan nanoparticles enhanced thickness of blade (µ), thickness of mesophyll tissue, thickness of the lower and upper epidermis and bundle length and width in the midrib compared to the control. In the control treatment's top epidermis, several sori and a large number of urediniospores were found. Most anatomical characters of flag leaves in control plants were reduced by biotic stress with P. triticina. Transcription levels of PR1-PR5 and PR10 genes were activated in chitosan nanoparticles treated plants at 0, 1 and 2 days after inoculation. In light of the data, we suggest that the prospective use of chitosan nanoparticles might be an eco-friendly strategy to improve growth and control of leaf rust disease.

Biological and Molecular Characterization of a Jumbo Bacteriophage Infecting Plant Pathogenic Ralstonia solanacearum Species Complex Strains.

A jumbo phage infecting Ralstonia solanacearum species complex strains, designated RsoM2USA, was isolated from soil of a tomato field in Florida, United States, and belongs to the family Myoviridae. The phage has a long latent period of 270 min and completed its infection cycle in 360 min with a burst size of approximately 32 particles per cell. With a genome size of 343,806 bp, phage RsoM2USA is the largest Ralstonia-infecting phage sequenced and reported to date. Out of the 486 ORFs annotated for RsoM2USA, only 80 could be assigned putative functions in replication, transcription, translation including 44 tRNAs, and structure with the main structural proteins experimentally confirmed. Phylogenetic analyses placed RsoM2USA in the same clade as Xanthomonas phage XacN1, prompting a proposal of a new genus for the two jumbo phages. Jumbo phage RsoM2USA is a lytic phage and has a wide host range, infecting each of the three newly established Ralstonia species: R. solanacearum, R. pseudosolanacearum, and R. syzygii, and significantly reduced the virulence of its susceptible R. solanacearum strain RUN302 in tomato plants, suggesting that this jumbo phage has the potential to be developed into an effective control against diseases caused by R. solanacearum species complex strains.

A new Streptomyces scabies-infecting bacteriophage from Egypt with promising biocontrol traits.

Potato common scab caused by Streptomyces scabies is one of the most economically important diseases infecting potato. It reduces the quality of potato tubers, which subsequently decreases the tuber prices and causes significant economic losses for potato growers. Biological control using bacteriophages is a promising strategy for controlling this disease. In this study, a novel bacteriophage with high lytic efficacy against S. scabies was isolated from a potato field at El-Minya, Egypt, and was designated SscP1EGY. The phage has an icosahedral head of 55 nm and a short tail of 7.5 nm, typical of a podovirus. Its infection cycle was 90 min, including 50 min of latent time and 40 min of rise period with a burst size of approximately 200 PFU per infected cell. The genome of SscP1EGY consists 51,751 nucleotides with 76 predicted genes. SscP1EGY infected and completely lysed seven tested S. scabies strains but showed no lytic activity against three beneficial Streptomyces species, other beneficial bacterial species, and non-target plant pathogenic bacteria. In greenhouse experiments, treatment of S. scabies-inoculated potato tubers with phage SscP1EGY resulted in reductions of (1) the severity of scab, (2) the number of lesions, and (3) the percentage of lesion surface, as compared to the inoculated tubers without phage treatment. Also, scab lesions appeared superficial in phage-treated tubers but pitted in non-phage-treated tubers. Our results suggest that SscP1EGY has a potential as a biological control agent for S. scabies. Based on our knowledge, SscP1EGY is the first sequenced S. scabies-infecting phage in Egypt.

Molecular and Biological Characterization of Ralstonia Phage RsoM1USA, a New Species of P2virus, Isolated in the United States.

The first Ralstonia-infecting bacteriophage from soil of the United States, designated RsoM1USA, was isolated from a tomato field in Florida. Electron microscopy revealed that phage RsoM1USA is member of the genus P2virus in the family Myoviridae with an icosahedral head of about 66 nm in diameter, a contractile tail of about 152 nm in length, and a long "neck." Phage RsoM1USA infected 12 of the 30 tested R. solanacearum species complex strains collected worldwide in each of the three Ralstonia species: R. solanacearum, R. pseudosolanacearum, and R. syzygii. The phage completed its infection cycle 180 min post infection with a burst size of about 56 particles per cell. Phage RsoM1USA has a genome of 39,309 nucleotides containing 58 open reading frames (ORFs) and is closely related to Ralstonia phage RSA1 of the species Ralstonia virus RSA1. The genomic organization of phage RsoM1USA is also similar to that of phage RSA1, but their integrases share no sequence homology. In addition, we determined that the integration of phage RsoM1USA into its susceptible R. solanacearum strain K60 is mediated by the 3' 45-base portion of the threonine tRNA (TGT), not arginine tRNA (CCG) as reported for phage RSA1, confirming that the two phages use different mechanism for integration. Our proteomic analysis of the purified virions supported the annotation of the main structural proteins. Infection of a susceptible R. solanacearum strain RUN302 by phage RsoM1USA resulted in significantly reduced growth of the infected bacterium in vitro, but not virulence in tomato plants, as compared to its uninfected RUN302 strain. Due to its differences from phage RSA1, phage RsoM1USA should be considered the type member of a new species with a proposed species name of Ralstonia virus RsoM1USA.

Identification of a DNA region associated with the cool virulence of Ralstonia solancearum strain UW551 and its utilization for specific detection of the bacterium's race 3 biovar 2 strains.

The cool virulent Ralstonia solanacearum race 3 biovar 2 (r3b2) strains cause destructive brown rot of potato. They are quarantined pathogens in Europe and Canada and select agent pathogens in the United States. We previously identified r3b2 (sequevars 1 and 2)-unique fragments that clustered into 32 regions in the genome of R. solanacearum. In this study, we targeted five of those regions for mutagenesis in order to determine whether they are involved in cool temperature-related biological functions for diagnostic purpose. Knockout mutants of four regions produced no changes to the biology of the r3b2 strain UW551. The mutation of region 13, which is 3,407 bp in size, resulted in significantly reduced twitching motility, attachment to the roots of tomato seedlings, and virulence under cool temperature conditions (18-24°C), although no significant difference was found under warm temperature conditions (24-30°C) as compared to the wild type strain. As a result, we designed primer pair Rs-CV-F and Rs-CV-R to target the region 13 for specific detection of r3b2 strains of R. solanacearum. Our assay specifically detected all the 34 r3b2 strains and none of the 56 non-r3b2 strains of R. solanacearum, nor any other five plant- or soil-associated bacteria including Enterobacter cloacae, Pseudomonas syringae pv. syringae, Xanthomonas campestris pv. campestris, X. citri, and R. pickettii. Unexpectedly, in silico analysis predicted that a recently deposited non-sequevar 1 or 2 Brazilian R. solanacearum strain RS489 would be recognized by our assay and by previously published r3b2-specific assays, although the cool-virulent status of this strain is unclear. Our PCR assay is the first to target a DNA region associated with cool-virulence that makes r3b2 strains highly regulated pathogens for specific detection of this important group of R. solanacearum.

Host range and molecular characterization of a lytic Pradovirus-like Ralstonia phage RsoP1IDN isolated from Indonesia.

A lytic Ralstonia solanacearum-infecting phage designated Ralstonia phage RsoP1IDN was isolated from soil in Indonesia. The phage has a linear double-stranded DNA genome of 41,135 bp with 413-bp terminal repeats, and contains 41 annotated open reading frames. The phage is most closely related to Ralstonia phage RSB1, but different from RSB1 mainly in containing a putative HNH homing endonuclease and having a narrower host range. Our phylogenetic and genomic analyses revealed that both phages RsoP1IDN and RSB1 belong to the genus Pradovirus or a new genus, and not Phikmvvirus as previously reported for phage RSB1. RsoP1IDN is the first sequenced and characterized R. solanacearum-infecting phage isolated from Indonesia in the proposed species Ralstonia virus RsoP1IDN.

Sequencing, genome analysis and host range of a novel Ralstonia phage, RsoP1EGY, isolated in Egypt.

A novel Ralstonia phage was isolated from soil in Egypt. It was designated Ralstonia phage RsoP1EGY using our phage identifier naming approach to reflect the phage's bacterial host species, characteristics and origin. When tested, this phage specifically infected only race 3 biovar 2 phylotype IIB sequevar 1, and not non-race 3 biovar 2 strains of Ralstonia solanacearum. The phage has an icosahedral capsid of 60 ± 5 nm in diameter with a short tail of 15 ± 5 nm in length, typical of a podovirus. The genome of RsoP1EGY is 41,297 bp in size, containing 50 open reading frames, with no significant sequence identity to any other reported R. solanacearum or non-Ralstonia phages, except to the recently deposited but unreported and unclassified Ralstonia phage DU_RP_I. RsoP1EGY is the first sequenced and characterized R. solanacearum phage isolated in Egypt.

Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum.

A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum.

Prophage Rs551 and Its Repressor Gene orf14 Reduce Virulence and Increase Competitive Fitness of Its Ralstonia solanacearum Carrier Strain UW551.

We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum, indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant), and nine of the prophage's 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant), respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes (pilT, egl, pehC, hrPB, and phcA), and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a lysogenic phage and its carrier plant pathogenic bacterial strain by determining the effect of the prophage Rs551 and its repressor on the virulence and competitive fitness of its carrier strain UW551 of R. solanacearum.

The filamentous phage XacF1 causes loss of virulence in Xanthomonas axonopodis pv. citri, the causative agent of citrus canker disease.

In this study, filamentous phage XacF1, which can infect Xanthomonas axonopodis pv. citri (Xac) strains, was isolated and characterized. Electron microscopy showed that XacF1 is a member of the family Inoviridae and is about 600 nm long. The genome of XacF1 is 7325 nucleotides in size, containing 13 predicted open reading frames (ORFs), some of which showed significant homology to Ff-like phage proteins such as ORF1 (pII), ORF2 (pV), ORF6 (pIII), and ORF8 (pVI). XacF1 showed a relatively wide host range, infecting seven out of 11 strains tested in this study. Frequently, XacF1 was found to be integrated into the genome of Xac strains. This integration occurred at the host dif site (attB) and was mediated by the host XerC/D recombination system. The attP sequence was identical to that of Xanthomonas phage Cf1c. Interestingly, infection by XacF1 phage caused several physiological changes to the bacterial host cells, including lower levels of extracellular polysaccharide production, reduced motility, slower growth rate, and a dramatic reduction in virulence. In particular, the reduction in virulence suggested possible utilization of XacF1 as a biological control agent against citrus canker disease.

Characterization of bacteriophages Cp1 and Cp2, the strain-typing agents for Xanthomonas axonopodis pv. citri.

The strains of Xanthomonas axonopodis pv. citri, the causative agent of citrus canker, are historically classified based on bacteriophage (phage) sensitivity. Nearly all X. axonopodis pv. citri strains isolated from different regions in Japan are lysed by either phage Cp1 or Cp2; Cp1-sensitive (Cp1(s)) strains have been observed to be resistant to Cp2 (Cp2(r)) and vice versa. In this study, genomic and molecular characterization was performed for the typing agents Cp1 and Cp2. Morphologically, Cp1 belongs to the Siphoviridae. Genomic analysis revealed that its genome comprises 43,870-bp double-stranded DNA (dsDNA), with 10-bp 3'-extruding cohesive ends, and contains 48 open reading frames. The genomic organization was similar to that of Xanthomonas phage phiL7, but it lacked a group I intron in the DNA polymerase gene. Cp2 resembles morphologically Escherichia coli T7-like phages of Podoviridae. The 42,963-bp linear dsDNA genome of Cp2 contained terminal repeats. The Cp2 genomic sequence has 40 open reading frames, many of which did not show detectable homologs in the current databases. By proteomic analysis, a gene cluster encoding structural proteins corresponding to the class III module of T7-like phages was identified on the Cp2 genome. Therefore, Cp1 and Cp2 were found to belong to completely different virus groups. In addition, we found that Cp1 and Cp2 use different molecules on the host cell surface as phage receptors and that host selection of X. axonopodis pv. citri strains by Cp1 and Cp2 is not determined at the initial stage by binding to receptors.