Efficacy of Pseudomonas chlororaphis subsp. aureofaciens SH2 and Pseudomonas fluorescens RH43 isolates against root-knot nematodes (Meloidogyne spp.) in kiwifruit

The Root-knot nematodes, Meloidogyne spp., are parasites of many crops and orchards, including kiwifruit trees. The Islamic Republic of Iran is among the leading kiwifruit producers in the world and M. incognita has been found as the dominant species responsible for severe loss of this crop. In order to evaluate the eff ectiveness of antagonistic bacteria on larval mortality, number of galls per plant and egg masses of nematode reduction, fifty local bacterial strains were isolated from root surrounding soils of kiwifruit plants in the northern production areas in Iran. Bacterial antagonists were characterized by morphological, physiological, biochemical and molecular methods. Two representative strains, showing the best nematicidal activity, were identif ed as Pseudomonas chlororaphis subsp. aureofaciens (isolate Sh2) and Pseudomonas fluorescens (isolate Rh43). They increased the percentage of larval mortality to 56:38% and 54:28% respectively in assays in vitro and showed excellent performance also in vivo with consistent reduction of number of galls (67:31% and 55:63%, respectively) and egg mass (86:46% and 84:29%, respectively) in plants. This study indicates that Pseudomonas chlororaphis subsp. aureofaciens isolate Sh2 and Pseudomonas fluorescens isolate Rh43 are good potential biocontrol agents for containing root-knot nematodes in kiwifruit trees.peer-reviewe

The Viable but Non-culturable State in Xanthomonas citri subsp. citri is a Reversible State Induced by Low Nutrient Availability and Copper Stress Conditions

Xcc (Xanthomonas citri subsp. citri) causes citrus bacterial canker, a leaf, stem and fruit spotting disease that affects most commercial citrus species and cultivars. Copper compounds, widely used for management of this pathogen, have been reported as inducers of a VBNC (viable but non-culturable state) in plant pathogenic bacteria. VBNC may be considered as a state preceding bacterial death or as a survival mechanism under adverse conditions. Several experiments were performed to characterize the reversibility and persistence of the VBNC state in Xcc. VBNC was induced in low nutrient medium or with amendment of copper at concentrations used for field disease control. The VBNC condition was demonstrated to persist up to 150 days after copper treatment and was reversed after the addition of culture media without copper or amendment with citrus leaf extract. Xcc viability was evaluated by recovery of colonies on culture media, confirmed by membrane integrity, respiratory activity and by real-time RT-PCR targeting a sequence from the gumD gene. Besides, the colonies recovered were pathogenic on citrus leaves. These results confirm that the VBNC state in Xcc is inducible and reversible and therefore may occur in the phyllosphere when Xcc is under copper stress or starvation

Efficacy of Pseudomonas chlororaphis subsp. aureofaciens SH2 and Pseudomonas fluorescens RH43 isolates against root-knot nematodes (Meloidogyne spp.) in kiwifruit

The Root-knot nematodes, Meloidogyne spp., are parasites of many crops and orchards, including kiwifruit trees. The Islamic Republic of Iran is among the leading kiwifruit producers in the world and M. in- cognita has been found as the dominant species responsible for severe loss of this crop. In order to evaluate the effectiveness of antagonistic bacteria on larval mortality, number of galls per plant and egg masses of nematode reduction, fifty local bacterial strains were isolated from root surrounding soils of kiwifruit plants in the northern production areas in Iran. Bacterial antagonists were characterized by morphological, physiological, biochemical and molecular methods. Two representative strains, showing the best nematicidal activity, were identifed as Pseudomonas chlororaphis subsp. aureofaciens (isolate Sh2) and Pseudomonas fluorescens (isolate Rh43). They increased the percentage of larval mortality to 56.38% and 54.28% respectively in assays in vitro and showed excellent performance also in vivo with consistent reduction of number of galls (67.31% and 55.63%, respectively) and egg mass (86.46% and 84.29%, respectively) in plants. This study indicates that Pseudomonas chlororaphis subsp. aureofaciens isolate Sh2 and Pseudomonas fluorescens isolate Rh43 are good potential biocontrol agents for containing root-knot nematodes in kiwifruit trees

Phenotypic and Genotypic Evaluation of Pseudomonas syringae pv. syringae Strains, Causing Citrus Blast in the West of Mazandaran and the East of Guilan

Introduction: P. syringae pv. syringae (P.s.s), the causal agent of blast of citrus trees, is one of the most important plant pathogens in the world. P.s.s is unique among most P. syringae pathovars according to its ability to cause disease in over 180 species of plants in several unrelated genera. Traditionally, Strains of P.s.s are identified on the basis of biochemical and nutritional tests and symptom expression in host plants. Genomic fingerprinting methods based on the polymerase chain reaction (PCR) have been applied for identification and classification of plant-associated bacteria to the subspecies level. The objectives of this study were the phenotypic and molecular evaluation of P.s. pv. syringae strains causing citrus blast in the West of Mazandaran and the East of Guilan, and study of genetic diversity of P.s.s isolates of citrus by using ERIC and REP-PCR markers. Materials and Methods: During 2011 to 2012, citrus infected tissues were sampled from different orchards in the West of Mazandaran and the East of Guilan. Bacterial phenotypes were studied based on standard physiological and biochemical tests. Gram reaction was determined by potassium hydroxide solubility test (KOH test). Strains were grown on King'B medium (KB) and fluorescent pigment production was evaluated. Levan formation, oxidase reaction, potato soft rot, Arginine dihydrolase and induction of the hypersensitive reaction in tobacco leaves (LOPAT tests), were done as described by Schadd et al. The standard strains of P.s. pv. syringae form IVIA were used as reference strains in this study. Pathogenicity Test was done as described by Yessad et al. Citrus seedlings were maintained in a greenhouse at 20°C. In addition, a PCR-based method was used to confirm the genus and species of bacteria by using bacterial specific primer pair’s designed for a specific gene of syringomycin B. Genetic diversity among the strains, was studied by rep-PCR fingerprinting. Genomic fingerprinting was carried out according to the methods of rep-PCR with ERIC primers of Little et al. and with REP primers of Zhao et al. The amplified PCR products were analyzed by gel electrophoresis on a gel containing 1.5% agarose in 1 × TAE buffer. Similarity analyses were done with the NTSYSpc ver. 2.02 software (Exeter Software, New York, USA) as described by Rohlf. Similar coefficients were compared using SM coefficient analysis according to the number and position of bands. Dendrograms were produced according to the unweighted pair-group mean arithmetic method (UPGMA) using NTSYSpc software. Results and Discussion: Bacterial strains were identified on the basis of Phenotypic and pathogenicity tests. Twenty- seven isolates of bacteria identified as P. syringae pv. syringae. Strains in which the gram reaction was negative were investigated based on physiological and biochemical characteristics. All P.s.s strains used in this study were negative for oxidase, potato rot, and Arginine dihydrolase but, positive for levan production and the hypersensitive response in tobacco. Strains were positive in gelatin, casein, aesculin, fluorescent pigment production, and tolerant to 5% NaCl, syringomycin production tests, but negative for urease, hydrolysis of starch. Growth at 370C, reduction of nitrate to nitrite and the hydrolysis of tween 80 were variable. These strains produced acid from glucose, xylose, sorbitol, galactose, sucrose and mannitol, but did not produce acid from maltose, Ramnose and the use of lactose were variable. All of the P.s.s strains were pathogenic on seedlings and produced progressive necrotic symptom. To confirm biochemical identification of the bacterial strains, the specific markers were selected for PCR. All 27 isolates of the P.s.s and the standard strains of P.s.s form IVIA produced the expected 198 bp. fragment of the gene syrB with specific markers. The isolates were determinated as P. s. pv. syringae based on phenotypical features and molecular identification. Traditionally, strains of P.s.s are recognized based on biochemical, nutritional, and physiological characteristics of citrus in different parts of Mazandaran. To assess genetic diversity among the strains, ERIC and REP-PCR analysis were used. Strains formed 6 and 5 clusters in the ERIC-PCR and REP-PCR, at 75% similarity level respectively and by the combination data set of both ERIC and REP-PCR, strains formed 6 clusters. In addition, strains formed 2 clusters at 62% similarity level. Cluster one contained the strains of citrus from Kelarabad, Chaboksar, Ketalem. The second Cluster contained other strains with the standard strains of P.s.s (IVIA). Diversity among P.s.s strains using rep-PCR fingerprinting was considerable. The results of this study demonstrated that P.s.s strains isolated from citrus trees are genotypically heterogeneous. Conclusion: In Iran, P.s.s strains were isolated from citrus and characterized. They emphasized phenotypic and nutritional. Whereas genotypic features of this pathogen have not been studied yet. In our study, according to phenotypic and molecular methods, strains were identified as P. s. pv. syringae. Despite similar phenotypic characteristics, the results indicated the existence of genetic heterogeneity among Pss strains causing citrus blast in the West of Mazandaran and the East of Guilan. The rep-PCR method is low cost, rapid, and reliable to discriminate plant-pathogenic bacteria at the pathovar level. Similar results have been reported in other studies of the strains isolated from other plants. Disclosing the population diversity of each pathovar, in turn, has implications for the implementation of breeding programs, disease management strategies, and ecological and epidemiological studies

Reaction of Commercial Cultivars of Kiwifruit to Infection by Root-knot Nematode and Its Biocontrol Using Endophytic Bacteria

Root-knot nematodes (RKN) cause considerable economic losses to kiwifruit production annually. Screening of resistant cultivars has been one of the long-standing methods to manage root-knot nematodes. Here, the reaction of the four most common commercial cultivars of kiwifruit, namely, Actinidia chinensis var. deliciosa cv. Hayward, A. chinensis var. deliciosa cv. Abbott, A. chinensis var. deliciosa cv. Bruno, and A. chinensis var. chinensis cv. Haegeum (commonly known as ‘Golden’ kiwifruit) to infection by the RKN, Meloidogyne incognita, was evaluated. Among examined cultivars ‘Golden’ was the most susceptible, having on average 52.8 galls, 56.1 egg masses per gram of root, and 642 J2 population per 200 gram of soil. ‘Bruno’ showed the highest resistance, with 3.3 galls, 4.1 egg masses per gram of root, and 79 J2 in 200 g of soil. Then, two potential biological control agents, namely Priestia megaterium 31.en and Agrobacterium tumefaciens 19.en were used on ‘Hayward’ seedlings against M. incognita and showed a significant reduction in the number of galls and egg masses on roots, juvenile population in the soil, and increased the growth parameters of the plants compared to non-treated seedlings. We demonstrated that integrated management using resistant cultivars and biological control can provide a safe and economic method to control RKN, and these resistant cultivars can be used in breeding programs

Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp citri under different treatments

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate

mRNA from selected genes is useful for specific detection and quantification of viable Xanthomonas citri subsp citri

The purpose of this study was to assess the stability of mRNA and rRNA for evaluation of viability for Xanthomonas citri subsp. citri (Xcc). Total RNA from Xcc suspensions subjected to different stress treatments (high temperature or chemical treatment with sodium orthophenylphenate at different concentrations) was extracted at different time periods post-treatment (0, 3, 24 and 48 h) and analysed by quantitative real-time reverse transcription PCR (Q-RT-PCR). Primers were designed from selected fragments of rRNA and mRNA from genes involved in bacterial fitness, virulence or general metabolic mechanisms (gumD, rpfB, avrBs2 and gyrB). After stress treatment, only a 445-bp fragment from the gumD mRNA was detected in live Xcc cells specifically, whereas other RNA fragments, as well as DNA targets, were detected in both viable and nonviable cells. Statistical analyses demonstrated that the amount of some transcripts from genes involved in xanthan synthesis, pathogenicity factor regulation and DNA processing was significantly reduced after lethal treatments. The amplification of the 445-bp product from gumD mRNA was demonstrated to be useful for the detection of viable Xcc; the product was detected specifically from viable bacteria on leaf and citrus fruit surfaces and in citrus canker lesions. Instability of long RNA fragments can be used as a practical tool for the study of survival of citrus canker bacteria or for diagnostic purposes when the presence of viable bacteria needs to be confirmed

Diagnosis of Xanthomonas axonopodis pv. citri, causal agent of citrus canker, in commercial fruits by isolation and PCR-based methods

Aims: To show the results of the detection of an EU quarantine organism, Xanthomonas axonopodis pv. citri (Xac), in citrus fruits imported from countries where this bacterium is present, using an integrated approach that includes isolation, pathogenicity assays and molecular techniques. Methods and Results: Citrus fruits with canker-like symptoms, exported to Spain from South American countries were analysed by several methods. Bacterial isolation, three conventional polymerase chain reaction (PCR) protocols, and real-time PCR with SYBR Green or a TaqMan probe, were compared. Canker-like lesions were disrupted in PBS buffer, and the extract used for bacterial isolation and DNA extraction followed by PCR amplification. Canker lesions, identified by PCR, showed viable bacteria in eleven of fifteen fruit samples. In 16 out of 130 lesions analysed from these samples, Xac was isolated, and pathogenicity on grapefruit leaves confirmed. By real-time PCR, using SYBR green or a Taqman probe, Xac was detected in 58 and 80 lesions respectively. By conventional PCR the bacterium was detected in 39–52 lesions depending on the protocol employed. Conclusions: An integrated approach for reliable detection of Xac in lesions of fruit samples, employing several techniques and with real-time PCR using a TaqMan probe as the fastest and most sensitive screening method, has been established and validated and is proposed as a useful tool for the analysis of Xac on fresh fruits. Significance and Impact of the Study: This work faces up to the real threat of the importation of citrus fruits that can harbour quarantine bacteria and will be useful in diagnostic laboratories for the analysis of commercial fresh fruits from countries where citrus canker is present

Land suitability procedure for sustainable citrus planning using the application of the analytical network process approach and GIS

Land use planning and ecological land evaluation are considered the most important tools and factors of sustainable development. Two aspects are of importance, firstly the potential suitability of the land for a specific use and the secondly management practices that integrate various factors such as agro-ecological aptitude, environmental impact, hydro-climate conditions and socio-economic constraints. The aim of this paper is to identify the variety of interactions, dependencies and feedback between higher and lower level factors, and the impact of these interacting factors on sustainable citrus production. This new framework incorporates three-discipline criteria: socio-economic status, topography and hydro-climate. In this proposed multi-criteria model, the Analytic Network Process (ANP) enabled us to consider interdependency among the 14 different criteria. Based on experts' opinion weights were assigned to each of these 14 different criteria and using the ANP and GIS-MCDM, potential areas based on the most important, or limiting factors were determined. The results of this land suitability procedure (LSP) indicate a number of critical factors, which would help managers to achieve optimum crop yield and decrease the loss of citrus production. According to experts' opinion, higher weights were assigned to minimum temperature and altitude than to all other criteria. The results also demonstrate that climate conditions, and topography play a major role in potential citrus expansion. Suitable regions (free risk areas) for citrus production were identified based on major environmental factors and an optimum suitability map was obtained by overlaying 14 GIS layers. This map will be of value for future citrus planning decisions; and could lead to reduction in citrus investment and expansion into high-risk area