Phylloplane as a potential Erwinia psidii inoculum reservoir

Essential oils (EOs) are natural products widely used in sustainable agrochemistry, not only because they are biodegradable and safe but also because they are regarded as alternatives to chemical fungicides against fungal species that attack crops. Allelopathy, another field of study, falls within the most recent and sustainable strategies applied to weed suppression to replace synthetic herbicides. Therefore, this study reports the chemical composition and allelopathic and antifungal effects of the EOs extracted from Calyptranthes concinna dried leaves (Cc-EO) and its pure major constituent elemicin. Their antifungal activities were evaluated by the disk diffusion method (DDM) at doses between 0.05 mg/mL and 0.4 mg/mL of Cc-EO and elemicin. The allelopathic effect was evaluated by studying the inhibition of germination and the growth of Lactuca sativa seeds. The chemical composition of Cc-EO was determined by GC-MS and GC-FID analyses. The major constituents of Cc-EO were elemicin (60.5%), α-cadinol (9.0%) and caryophyllene oxide (8.3%). Cc-EO and elemicin were assayed in vitro against 17 fungi of agronomic interest (Aspergillus niger, A. flavus, A. nomius, Penicillium digitatum, P. expansum, Sclerotinia sclerotiorum, S. rolfsii, S. minor, Fusarium graminearum, Myrothecium verrucaria, Corynespora cassiicola, Erwinia psidii, Colletotrichum musae, Alternaria carthami, Rhizoctonia solani, Rhizopus stolonifer and Macrophomina phaseolina). The concentration of Cc-EO (0.4 mg/mL) inhibited 100% of the mycelium growth of seven strains, equal to the fungicide fluazinam, which was used as a positive control. Elemicin showed antifungal activity against all fungi at all concentrations under investigation (above 50%). A strong allelopathic effect was recorded for Cc-EO and elemicin at the dose of 0.28 mg/mL, with the almost total inhibition of germination. This study revealed, for the first time, the strong and remarkable fungicidal and allelopathic effects of Cc-EO and elemicin, an important finding for the agrochemical field.

Comprehensive prediction of plant cytoplasmic and apoplastic effectors underlying <i>Erwinia psidii</i> pathogenicity

Abstract Erwinia psidii is an airborne bacterium that is the causal agent of eucalyptus bacterial blight in Brazil. However, there are no studies on the survival of the bacterial pathogen under epiphytic, soil and eucalyptus leaf debris conditions. We investigated the survival of E. psidii on the phylloplane and in leaf debris from two eucalyptus genotypes, and within eucalyptus forest soil. A rifampicin‐resistant mutant (CR01R) was obtained from the wild type by the gradient plate method and chosen for the study because it showed the same morphological, growth, pathogenic and aggressive properties as the parental strain. On the phylloplane of Eucalyptus urophylla × Eucalyptus globulus and E. saligna, the plant–pathogenic bacterium was recovered for up to 40 and 60 days, respectively. Under non‐sterile eucalyptus forest soil, E. psidii survived for 15 days post–soil infestation. In leaf debris from the eucalyptus genotypes, the bacterial pathogen was detected for up to 90 days in debris placed on the soil surface. However, under non‐sterile soil, 5 or 15 cm deep, the bacterium was detected for up to 45 and 60 days in leaf debris from E. saligna and E. urophylla × E. globulus, respectively. In this study, we showed that E. psidii can survive as an epiphyte or saprophyte in diseased leaves on the soil. However, the pathogen was a poor survivor in natural eucalyptus forest soil. The pathogen's survival period in leaf debris buried in the soil was drastically reduced. This information shows that the phylloplane or leaf debris is putative reservoirs for the bacterium and may serve as a primary inoculum source.

Water quality analysis is essential in assessing the suitability of different water sources used for various purposes, including industrial and domestic uses. This study was therefore aimed at determining the physicochemical and bacteriological quality of water samples from different storage facilities in a tertiary institution in Port Harcourt, Rivers State, Nigeria. The study involved eighty (80) water samples obtained from water storage tanks situated at 16 locations within the premises of the institution, to ascertain the physicochemical property, presence and population of different bacterial groups influencing the quality of these water sources. In-situ and ex-situ physicochemical analyses as well as bacteriological investigations were carried out on all samples, using standard laboratory procedures. Results of physicochemical analysis showed that the pH ranged from 4.15±0.14 to 7.16±0.08; conductivity, from 50.55±0.49 (µs/cm) to 364.00±2.83; salinity, from 0.02±0 (ppt) to 0.18±0; temperature, from 27oC to 28oC; Chloride, from 1.03±0.06 (mg/l) to 10.80±0.79; total alkalinity, from 4.00±0 (mg/l) to 11.00±1.41; Dissolved oxygen from 3.04±.020 to 7.36±0.08 (Mg/l) and BOD ranged from 0.81±0 to 4.23±0.09 (Mg/l). Results for bacterial population showed total heterotrophic bacteria ranging from 1.03±1.19 x 103 CFU/ml in water from reservoir tanks at the Faculty of Engineering, to 5.89±2.59 x 103 CFU/ml at Road A Block B; total coliform count ranged from 0 CFU/ml in most samples, to 10.00±2.36 x 102 CFU/ml in Block B reservoir tanks. Water reservoirs in clinic area recorded the highest Salmonella/Shigella counts (1.00±0.23 x 101 CFU/ml) with other stations having zero counts. Hall F (Hostel Ext) on the other hand had the highest counts for Vibrio (2.20±3.01 x 101 CFU/ml). The phenotypic characterization identified Citrobacter spp. as the most occurring (27.27%) bacterial isolate in the study, followed by Alcaligenes faecalis and Klebsiella spp. (18.18% each). Enterobacter spp., Edwardsiella spp., Erwinia psidii, Acinetobacter spp., Pseudomonas spp., Providentia spp. and Salmonella spp. all appeared as the least occurring, having a percentage of 4.55%, each. Tatumella spp. on the other hand had a percentage occurrence of 9.09%. This study has buttressed the need for increased water hygiene of reservoir tanks as well as water sources in these locations.

Improved evaluation of the genetic variability of Brazilian strains of <i>Erwinia psidii</i> with newly developed microsatellite markers

Prediction, structure characterization, and evolutionary analysis of <i>Erwinia psidii</i> putative type III effectors

Bidirectional colonization and biofilm formation by <i>Erwinia psidii</i> in eucalypt plants

Abstract Dieback and wilt, caused by Erwinia psidii (Ep), is one of the most important emergent diseases of Eucalyptus spp. in Brazil. Currently, pathogen detection relies on isolation of bacteria from infected plant tissue and either identification based on morphological, physiological and biochemical tests or DNA amplification using the polymerase chain reaction (PCR), which in many cases is laborious and cumbersome. Considering the need for a simpler and more rapid, yet reliable, method for detecting the pathogen, we obtained a polyclonal antibody (anti‐Ep) and developed an agglutination test for specific detection of E. psidii. The antiserum was produced against the E. psidii strain LPF534 and tested against 101 E. psidii isolates from Eucalyptus spp.; three E. psidii isolates from Psidium guajava; 23 Ralstonia solanacearum and 18 Xanthomonas axonopodis isolates pathogenic to Eucalyptus spp.; and seven endophytic isolates from Eucalyptus spp., three of which are phylogenetically related to the genus Erwinia. Results of direct ELISA indicated that a concentration as low as 3.5 µg/ml of the anti‐Ep antibody was able to detect the E. psidii antigen and that the antibody did not cross‐reacted with other bacteria pathogenic and non‐pathogenic to Eucalyptus spp. In the agglutination test, the anti‐Ep antibody showed positive reaction with all strains of E. psidii tested whereas cross‐reaction with none of the strains that belong to other taxonomic groups was observed. The agglutination test showed a detection limit of 105 colony‐forming units (CFU)/ml, and its specificity was the same as that obtained by PCR amplification using E. psidii‐specific primers. These results demonstrate that the agglutination test developed here is a useful tool for specific, fast and inexpensive detection of E. psidii although only operational on pure bacterial suspensions and not yet directly from infected tissues.

Aims: Erwinia psidii was first reported in 2017 to be the causal pathogen of papaya dieback disease (PDD) in Sabah, Malaysia. The present study aimed to isolate potential biocontrol agents against this pathogen. Methodology and results: Out of the 20 samples collected from Crocker Range of Sabah, 154 bacteria and 55 fungi isolates were isolated and screened for their antagonistic activity against E. psidii. The fungi and bacteria which gave the highest inhibition to E. psidii were identified using molecular technique as Alcaligenes faecalis and Lecanicillium sp., respectively. Both isolates were selected for extraction of their secondary metabolites to determine their bioactivity against E. psidii. Micro-well dilution method was used to determine minimum inhibitory concentration (MIC) for each microbes' extract. GC-MS analysis was carried out to determine their secondary metabolites. Conclusion, significance and impact of study: Lecanicillium sp. (Diethyl ether, chloroform and ethyl acetate) extracts and A. faecalis (Diethyl ether extract) showed positive inhibition against E. psidii. GC-MS analysis revealed that both A. faecalis and Lecanicillium sp. had secreted some secondary metabolites such as N-formylmaleamic acid, Oleamide and D-1-Piperideine-2-carboxylic acid that may relate to the growth inhibition. Alcaligenes faecalis and Lecanicillium sp. are potential to be further investigated as biocontrol against E. psidii.

Abstract Dieback caused byErwinia psidiiis currently one of the most important emerging diseases in eucalypt plantations in Brazil. However, little is known in terms of the host range of this pathogen or the potential sources of resistance against the disease it causes. In this study, we inoculated plants of species from nine families to gain insight into the host range ofE. psidii. Plants of all inoculated species of Myrtaceae exceptAcca sellowianaexhibited disease symptoms and therefore represent potential hosts for the pathogen under natural conditions. In addition, the response of fourCorymbiaspecies, 29Eucalyptusspecies and three interspecificEucalyptushybrids to inoculation withE. psidiiwas evaluated. AllCorymbia henryi,Corymbia maculata,Eucalyptus thozetiana,Eucalyptus cloeziana,Eucalyptus viminalis,Eucalyptus dalrympleanaandEucalyptus pilularisplants were highly resistant to the pathogen, whereas differential disease resistance was observed in the other species. This study provides important information on sources of resistance toErwinia psidiiwith potential use in the development of clones with enhanced resistance in eucalypt species of economic importance.

Erwinia psidii causes bacterial blight of guava, an important disease of this fruit crop in Brazil. Pathogen spread is associated with the movement of infected but asymptomatic propagating plant material. The objective of this study was to compare bacterial movement inside the host tissue and symptom development in two guava varieties commonly grown in Brazil. Guava plants of Pedro Sato and Kumagai varieties were inoculated with strain IBSBF435 by deposition of a bacterial suspension droplet followed by puncture at the axils of the first fully expanded pair of leaves. Disease severity was assessed in the upper third part of the plant and stem fragments were cut at intervals of 1 cm above and below the inoculation point. Samples were collected at 0.5, 1, 2, 4, 6, 8, 10 and 12 days after inoculation. Presence of viable cells of E. psidii at each sampling point was evaluated using Bio-PCR with species-specific primers after a 24 h-enrichment step. Bacterial movement was detected on both directions in both varieties, however a positive correlation between movement and disease severity was observed only for Pedro Sato. Kumagai plants remained symptomless despite the presence of E. psidii above and below the inoculation site.

Genetic diversity and aggressiveness of <i>Erwinia psidii</i> on <i>Eucalyptus</i> spp. in Brazil

ABSTRACT: Erwinia psidii causes bacterial blight of guava ( Psidium guajava ), an important disease of this crop in Brazil. The pathogen affects branches and twigs of guava trees, reducing yield significantly. Bacterial dissemination often occurs through contaminated but asymptomatic propagating plant material. The objectives of this research were to evaluate the use of BIO-PCR and conventional PCR to detect E. psidii in inoculated guava plants grown in a greenhouse and in symptomatic and asymptomatic trees from guava orchards. Erwinia psidii strain IBSBF 1576 was inoculated (107CFU mL-1) into young guava shoots and plant tissue was analysed at 0, 5, 10, and 15 days after inoculation. Symptoms were observed after 5 days and all inoculated shoots were PCR positive at all times, by both BIO-PCR and conventional PCR. Under natural infection conditions, 40 samples were tested by BIO-PCR from each of three guava orchards, 20 showing symptoms and 20 asymptomatic. PCR was positive for 58 out of 60 symptomatic samples (96.7%) and for 6.7% of asymptomatic samples, showing that the method can be used to detect the pathogen at early stages of infection. This PCR method may be used as a diagnostic tool to assess bacterial survival, dissemination and disease outbreaks.

ABSTRACT In this work, two cultural production systems were compared [conventional (CO) and organic (OR)], and its effects in the guava trees (Psidium guajava) bacterial blight (Erwinia psidii) control. The experimental design was in radomized blocks, in split-split-plot arrangement, where it was measured the bacterial disease and the fruits production on the 2005/06, 2006/07 and 2007/08 harvests. Four pruning seasons effects were evaluated on the harvests (September, December, March and June) in both production systems. Such systems were constituted of: OR – treatment with bioactive compound (BC), liquid BC and dead coverage, and; CO – chemical fertilization, fungicide and herbicide. In 2007/08, the area under the disease progress curve (AUDPC) of all the treatments in the OR system was lower (~54-107) than the CO one(~233-298). In the 2007/08 harvest the number of fruits for each plant for all the OR treatments was higher (~146-204) than the CO ones (~57-103). In all the harvests, considering all the treatments within each system, there was a significantly lower AUDPC (~93-184) and higher fruits production (~158-188) in the OR one than the CO one (AUDPC: ~208-476; fruits ~18-104). The pruning induced a higher AUDPC and lower fruits production in both production sytems.

Erwinia psidii causes bacterial blight of guava (Psidium guajava), one of the most important diseases of this crop in Brazil. Control measures are not effective, and dissemination often occurs through contaminated but asymptomatic propagating plant material. Considering the need for a reliable and sensitive method for detecting the pathogen in asymptomatic plant material, E. psidii-specific PCR primers were designed from a 355-bp fragment of the recombinase A gene (recA) amplified from E. psidii type strain. Primer pair Ep2L/2R only amplified DNA from E. psidii and its detection limit was 10−5 ng/μL of purified DNA and 10 CFU (colony forming units) of bacterial cell suspension/mL. Three methods, conventional PCR, IC-PCR, and BIO-PCR were evaluated with the selected primers for their potential to detect E. psidii on guava leaves. BIO-PCR and conventional PCR were more sensitive and less time-consuming than IC-PCR. The detection limits on extracts of macerated guava leaves spiked with bacterial suspensions at different concentrations were 10 and 103 CFU/mL for BIO-PCR and conventional PCR, respectively. The PCR method here described could be useful for developing a protocol for early detection of this pathogen in asymptomatic guava plants.

Erwinia psidii causes bacterial disease of guava in Brazil. Phenotypic and molecular characterization through rep-PCR fingerprinting of 42 strains from different geographical regions showed that E. psidii populations in Brazil have a low level of genetic diversity and suggest that contaminated plant material is the main source for pathogen dissemination in the country.

One of the most important diseases affecting guava production in Brazil is bacterial blight, caused by the bacterium Erwinia psidii. Pathogen dissemination often occurs through contaminated propagating plant material. The development of more effective diagnostic methods may reduce pathogen dissemination in the country. Considering the need for a reliable and simple method for detecting the pathogen in plant material, the objectives of this study were to produce E. psdii-specific polyclonal antibodies and to develop a detection method using bacterial population enrichment on guava leaf extracts followed by double radial immunodiffusion. The antiserum was produced against the E. psidii type strain IBSBF 435 (ICMP 8426, NCPPB 3555) and its efficiency, specificity and sensitivity threshold were determined. The antiserum As15-1 was tested with strains of several plant-pathogenic bacteria and reacted positively with all strains of E. psidii, although cross reactions were detected with two non-pathogenic isolates from guava flora. Bacterial multiplication on leaf extracts was observed 12 h after incubation from initial populations of 103, 105 and 107 ufc/mL, up to 60 h. After 12 h it was already possible to detect E. psidii in samples with starting populations of 107 cfu/mL. After 36 h, the enrichment technique allowed the detection of E. psidii using double radial immunodiffusion in samples with populations as low as 103 cfu/mL. Considering the possibility of false positives it is desirable to associate other diagnostic methods with the method proposed in this study.

The phytopathogen Erwinia psidii R. IBSBF 435T causes rot in branches, flowers, and fruits of guava (Psidium guajava L.), being responsible for crop losses, and has no effective control. It was demonstrated that this strain produces two compounds [S-(-)-N-hexanoyl and N-heptanoyl-homoserine lactone], both belonging to the class of quorum-sensing signaling substances. A protocol using gas chromatography-flame ionization detection with chiral stationary phase is described for the absolute configuration determination of a natural acyl-homoserine lactone. Biological assays with specific reporter and synthesis of identified substances are also described. This is the first report on the N-heptanoyl-homoserine lactone occurrence in the Erwinia genus.