Efficacy of different copper compounds in the control of Xanthomonas citri subsp. citri pathotypes A and A*

Citrus canker disease is one of the most devastating diseases that attacks citrus, especially limes in the Southern parts of Iran, and is caused by Xanthomonas citri subsp. citri (Xcc). The efficacy of several formulations of copper compounds including Bordeaux mixture, copper oxychloride and copper sulphate in controlling Xcc in Key lime was estimated in vitro and in planta using artificial inoculation. Specific primers were used to detect copper-resistant genes copA, copB and copL in 30 isolates of Xcc. The copA and copL genes were present in all isolates, and copB was detected only in 6 strains. In this study, we observed a very good in vitro growth inhibition activity of copper compounds against Xcc pathotype A. S14 strain (pathotype A^*) was the sole isolate that grew on media amended with 2/4 mM of Bordeaux mixture, copper oxychloride and copper sulphate. All other strains (pathotype A) failed to grow on media amended with this concentration. Bordeaux mixture exhibited high efficacy in controlling Xcc in both conditions. However, there were no significant differences in the efficacy of copper oxychloride and copper sulphate at 1.2 mM concentration in planta. A significantly minimum canker necrotic spot and highest disease control was achieved with Bordeaux mixture and copper oxychloride. There was a significant difference in disease severity of the type strain LMG9322 (pathotype A) and Xcc strain S14 (pathotype A^*). Our experiments showed that Bordeaux mixture exhibited satisfactory efficacy in controlling the causal agent of citrus canker.     

Evaluation of the antibiotic activity of extracellular compounds produced by the Pseudomonas strain against the Xanthomonas citri pv. citri 306 strain

In this work, we evaluated the antibiotic activity of metabolites produced by the Pseudomonas sp. LV strain and their effects on the cell morphology of the Xanthomonas citri pv. citri 306 strain (Xcc 306), which causes citrus canker lesions. The LV strain was cultivated, centrifuged, a cell-free supernatant was treated with dichloromethane and then concentrated, frozen in liquid nitrogen and lyophilized. The dichloromethane phase (DP) was fractionated by vacuum liquid chromatography (VLC) using six organic solvents with a crescent polarity. The antibiotic activity of the DP and all the fractions from VLC were tested against Xcc 306 and only the F3 fraction showed antimicrobial activity. The antibiotic activity of F3 was determined by minimum inhibitory concentration and the action on the cell morphology of Xcc 306 carried out in glass tubes with cell suspensions plus F3 solution sampled at three different times (one, three and six hours). The effects were analyzed by electron microscopy. Both the DP and F3 showed antibiotic activity against Xcc 306 in in vitro experiments. Electron microscopy showed that the F3 fraction completely disrupted the cell integrity after six hours. In a greenhouse experiment, the DP and F3 fraction (highly effective in in vitro experiments), reduced the formation of lesions by approximately 80% and 94%, respectively.     

The role as inoculum sources of Xanthomonas citri pv. citri surviving on the infected Satsuma mandarin fruits

Importing citrus fruits infected by Asiatic citrus canker caused by Xanthomonas citri pv. citri (Xcc) can act as an inoculum source for the disease epidemic in citrus canker-free countries. In this study, the pathogenicity of the causal agent of Asiatic citrus canker surviving on infected Satsuma mandarin fruits was evaluated. The washing solution of infected Satsuma mandarin fruits did not cause lesion formation on the citrus leaves. However, a typical citrus canker lesion was formed on the leaves after inoculation with higher concentrations of the inoculum from the washing solution (washing solution II). It indicated that the pathogenicity of the citrus canker surviving on the symptomatic Satsuma mandarin fruits was not changed. Scanning electron microscopic observation showed that the numbers of bacterial cells on the leaves of Satsuma mandarin which inoculated with the washing solution directly (washing solution I) was less compared to those of leaves inoculated with the washing solution II. This result spports that the pathogenicity of Xcc surviving on Satsuma mandarin fruits may not be changed but that the sucessful infection of citrus caker may depend on the concentration of the inoculum.     

Antibacterial Activity of Alkyl Gallates against Xanthomonas citri subsp. citri

The plant-pathogenic bacterium Xanthomonas citri subsp. citri is the causal agent of Asiatic citrus canker, a serious disease that affects all the cultivars of citrus in subtropical citrus-producing areas worldwide. There is no curative treatment for citrus canker; thus, the eradication of infected plants constitutes the only effective control of the spread of X. citri subsp. citri. Since the eradication program in the state of São Paulo, Brazil, is under threat, there is a clear risk of X. citri subsp. citri becoming endemic in the main orange-producing area in the world. Here we evaluated the potential use of alkyl gallates to prevent X. citri subsp. citri growth. These esters displayed a potent anti-X. citri subsp. citri activity similar to that of kanamycin (positive control), as evaluated by the resazurin microtiter assay (REMA). The treatment of X. citri subsp. citri cells with these compounds induced altered cell morphology, and investigations of the possible intracellular targets using X. citri subsp. citri strains labeled for the septum and centromere pointed to a common target involved in chromosome segregation and cell division. Finally, the artificial inoculation of citrus with X. citri subsp. citri cells pretreated with alkyl gallates showed that the bacterium loses the ability to colonize its host, which indicates the potential of these esters to protect citrus plants against X. citri subsp. citri infection.     

贝莱斯芽孢杆菌Bv-303对水稻白叶枯病菌的拮抗活性及其应用

本研究明确了一株新型贝莱斯芽孢杆菌(Bacillus velezensis) Bv-303菌株对黄单胞杆菌水稻致病变种(Xanthomonas oryzae pv. oryzae,Xoo)的拮抗活性及其对水稻白叶枯病(bacterial-blight,BB)的生物防治效果。采用牛津杯法测定了菌株Bv-303发酵上清液(cell-free supernatant, CFS)对白叶枯病菌体外拮抗的活性及其稳定性;通过对接种白叶枯病菌的水稻叶片进行喷雾处理,在水稻体内测试了该菌株发酵液(cell-culture broth,CCB)、发酵上清液及菌悬液(cell-suspension water,CSW)对白叶枯病菌的抑制效果;并统计了该菌株对水稻种子发芽率与幼苗生长的影响。结果表明,在体外,菌株Bv-303发酵上清液对白叶枯病菌的生长抑制率可达85.7%–88.0%,对热、酸、碱、紫外线等具有较好的稳定性;在水稻叶片上,喷施该菌株的发酵液、发酵上清液及菌悬液均能提高植株对白叶枯病的抗性,其中发酵液的效果最佳,抗病性提高率高达62.7%;且发酵液对水稻种子萌发和幼苗生长均没有副作用。因此,菌...     

Cyclic Lipopeptide Biosynthetic Genes and Products,and Inhibitory Activity of Plant-Associated Bacillus against Phytopathogenic Bacteria

The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP) biosynthetic genes ituC (iturin), bmyB (bacillomycin), fenD (fengycin) and srfAA (surfactin), and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP) genes were bmyB, srfAA and fenD (34-50% of isolates). Most isolates (98.4%) produced surfactin isoforms, 90.6% iturins and 79.7% fengycins. The antibacterial activity was very frequent and generally intense among the collection of strains because 75% of the isolates were active against at least 6 of the 8 bacterial plant pathogens tested. Hierarchical and correspondence analysis confirmed the presence of two clearly differentiated groups. One group consisted of Bacillus strains that showed a strong antibacterial activity, presented several cLPs genes and produced several isoforms of cLPs simultaneously, mainly composed of B. subtilis and B. amyloliquefaciens, although the last one was exclusive to this group. Another group was characterized by strains with very low or none antibacterial activity, that showed one or none of the cLP genes and produced a few or none of the corresponding cLPs, and was the most heterogenous group including B. subtilis, B. licheniformis, B. megaterium, B. pumilus, B. cereus and B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillus against plant pathogenic bacteria is related to the presence of cLP genes and to the production of the corresponding cLPs, and it is mainly associated to the species B. subtilis and B. amyloliquefaciens. Our findings would help to increase the yield and efficiency of screening methods to obtain candidate strains to biocontrol agents with a mechanism of action relaying on the production of antimicrobial cLPs.     

Lactose consuming strains of Xanthomonas citri subsp. citri (Xcc) insight into the emergence of natural field resources for xanthan gum production

Xanthomonas genus possesses a low level of β-galactosidase gene expression and is therefore unable to produce xanthan gum in lactose-based media. In this study, we report the emergence of some natural field strains of Xanthomonas citri subsp. citri (Xcc) capable to use lactose as a sole carbon source to produce xanthan gum. From 210 Xcc strains isolated from key lime (C. aurantifolia), 27 showed the capacity to grow on lactose containing medium. Xcc lactose consuming strains demonstrated a good level of xanthan production. Amongst all, NIGEBK37 produced the greatest (14.62 g/l) amount of xanthan gum in experimental laboratory conditions. By evaluating the viscosity of the biopolymer at 25 °C, it was demonstrated that xanthan synthesized by strain NIGEBK37 has the highest viscosity (44,170.66 cP). Our results were indicative for the weakness of a commercial strain of Xanthomonas campestris pv. Campestris DSM1706 (Xcc/DSM1706) to produce xanthan in lactose containing medium.     

Recent advances in the understanding of Xanthomonas citri ssp. citri pathogenesis and citrus canker disease management

Taxonomic status : Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas citri ssp. citri (Xcc). Host range : Compatible hosts vary in their susceptibility to citrus canker (CC), with grapefruit, lime and lemon being the most susceptible, sweet orange being moderately susceptible, and kumquat and calamondin being amongst the least susceptible. Microbiological properties : Xcc is a rod‐shaped (1.5–2.0 × 0.5–0.75 µm), Gram‐negative, aerobic bacterium with a single polar flagellum. The bacterium forms yellow colonies on culture media as a result of the production of xanthomonadin. Distribution : Present in South America, the British Virgin Islands, Africa, the Middle East, India, Asia and the South Pacific islands. Localized incidence in the USA, Argentina, Brazil, Bolivia, Uruguay, Senegal, Mali, Burkina Faso, Tanzania, Iran, Saudi Arabia, Yemen and Bangladesh. Widespread throughout Paraguay, Comoros, China, Japan, Malaysia and Vietnam. Eradicated from South Africa, Australia and New Zealand. Absent from Europe.     

Xylan Utilization Regulon in Xanthomonas citri pv. citri Strain 306: Gene Expression and Utilization of Oligoxylosides

Xanthomonas citri pv. citri strain 306 (Xcc306), a causative agent of citrus canker, produces endoxylanases that catalyze the depolymerization of cell wall-associated xylans. In the sequenced genomes of all plant-pathogenic xanthomonads, genes encoding xylanolytic enzymes are clustered in three adjacent operons. In Xcc306, these consecutive operons contain genes encoding the glycoside hydrolase family 10 (GH10) endoxylanases Xyn10A and Xyn10C, the agu67 gene, encoding a GH67 α-glucuronidase (Agu67), the xyn43E gene, encoding a putative GH43 α-l-arabinofuranosidase, and the xyn43F gene, encoding a putative β-xylosidase. Recombinant Xyn10A and Xyn10C convert polymeric 4-O-methylglucuronoxylan (MeGX_n) to oligoxylosides methylglucuronoxylotriose (MeGX₃), xylotriose (X₃), and xylobiose (X₂). Xcc306 completely utilizes MeGX_n predigested with Xyn10A or Xyn10C but shows little utilization of MeGX_n. Xcc306 with a deletion in the gene encoding α-glucuronidase (Xcc306 Δagu67) will not utilize MeGX₃ for growth, demonstrating the role of Agu67 in the complete utilization of GH10-digested MeGX_n. Preferential growth on oligoxylosides compared to growth on polymeric MeGX_n indicates that GH10 xylanases, either secreted by Xcc306 in planta or produced by the plant host, generate oligoxylosides that are processed by Xyn10 xylanases and Agu67 residing in the periplasm. Coordinate induction by oligoxylosides of xyn10, agu67, cirA, the tonB receptor, and other genes within these three operons indicates that they constitute a regulon that is responsive to the oligoxylosides generated by the action of Xcc306 GH10 xylanases on MeGX_n. The combined expression of genes in this regulon may allow scavenging of oligoxylosides derived from cell wall deconstruction, thereby contributing to the tissue colonization and/or survival of Xcc306 and, ultimately, to plant disease.     

Evaluation of rhizospheric Pseudomonas and Bacillus as biocontrol tool for Xanthomonas campestris pv campestris

Xanthomonas campestris pv campestris (Xcc), causing black rot, is one of the most yield-limiting and destructive pathogens of cruciferous crops. The intention of this study was to evaluate the potential of rhizobacteria in black rot management. Fifty-four isolates from rhizosphere soil of Brassica campestris were screened against Xcc. Two isolates namely, KA19 and SE, with inhibition radius >11 mm were selected. The combined use of them produced an average inhibition zone of 18.1 ± 1.4 mm radius (P < 0.05). 16S rRNA gene sequencing and phylogenetic analysis identified KA19 and SE as the nearest homologs (>99.4%) of Pseudomonas aeruginosa and Bacillus thuringiensis, respectively. In greenhouse study, both isolates were effective (P < 0.05) in reducing black rot lesions compared to untreated control involving either a foliar spray or the combined seed soak and soil drench. However, the combined strains (KA19 + SE) were significantly more effective (P < 0.05) when the mode of application was combined seed and soil drench. The lipid content of seeds increased significantly with the application of these strains, especially with SE alone and in combination. After 9 weeks, the Xcc population was significantly lower in soil treated with combined strains (P < 0.05). KA19 produced extracellular siderophores, influenced by various carbon sources and identified as 4-hydroxy-2-nonyl-quinoline by NMR. In Bacillus SE, two antibacterial factors corresponding to autolysins (β-N-acetylglucosaminidase) and AHL-lactonases were established. This study would strengthen our understanding for application of different rhizobacteria with various active principles like Pseudomonas and Bacillus as ingredients of a biocontrol mixture.     

Spiralin Diversity Within Iranian Strains of Spiroplasma citri

The first-cultured and most-studied spiroplasma is Spiroplasma citri, the causal agent of citrus stubborn disease, one of the three plant-pathogenic, sieve-tube-restricted, and leafhopper vector-transmitted mollicutes. In Iranian Fars province, S. citri cultures were obtained from stubborn affected citrus trees, sesame and safflower plants, and from the leafhopper vector Circulifer haematoceps. Spiralin gene sequences from different S. citri isolates were amplified by PCR, cloned, and sequenced. Phylogenetic trees based on spiralin gene sequence showed diversity and indicated the presence of three clusters among the S. citri strains. Comparison of the amino acid sequences of eleven spiralins from Iranian strains and those from the reference S. citri strain GII-3 (241 aa), Palmyre strain (242 aa), Spiroplasma kunkelii (240 aa), and Spiroplasma phoeniceum (237 aa) confirmed the conservation of general features of the protein. However, the spiralin of an S. citri isolate named Shiraz I comprised 346 amino acids and showed a large duplication of the region comprised between two short repeats previously identified in S. citri spiralins. We report in this paper the spiralin diversity in Spiroplasma strains from southern Iran and for the first time a partial internal duplication of the spiralin gene.     

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.     

HrpM is involved in glucan biosynthesis,biofilm formation and pathogenicity in Xanthomonas citri ssp. citri

Xanthomonas citri ssp. citri (Xcc) is the causal agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. A biofilm‐deficient mutant was identified in a screening of a transposon mutagenesis library of the Xcc 306 strain constructed using the commercial Tn5 transposon EZ‐Tn5 <KAN‐2> Tnp Transposome (Epicentre). Sequence analysis of a mutant obtained in the screening revealed that a single copy of the EZ‐Tn5 was inserted at position 446 of hrpM, a gene encoding a putative enzyme involved in glucan synthesis. We demonstrate for the first time that the product encoded by the hrpM gene is involved in β‐1,2‐glucan synthesis in Xcc. A mutation in hrpM resulted in no disease symptoms after 4 weeks of inoculation in lemon and grapefruit plants. The mutant also showed reduced ability to swim in soft agar and decreased resistance to H ₂ O ₂ in comparison with the wild‐type strain. All defective phenotypes were restored to wild‐type levels by complementation with the plasmid pBBR1‐MCS containing an intact copy of the hrpM gene and its promoter. These results indicate that the hrpM gene contributes to Xcc growth and adaptation in its host plant.     

KatG,the Bifunctional Catalase of Xanthomonas citri subsp. citri,Responds to Hydrogen Peroxide and Contributes to Epiphytic Survival on Citrus Leaves

Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker. This bacterium is exposed to reactive oxygen species (ROS) at different points during its life cycle, including those normally produced by aerobic respiration or upon exposition to ultraviolet (UV) radiation. Moreover, ROS are key components of the host immune response. Among enzymatic ROS-detoxifying mechanisms, catalases eliminate H₂O₂, avoiding the potential damage caused by this specie. Xcc genome includes four catalase genes. In this work, we studied the physiological role of KatG, the only bifunctional catalase of Xcc, through the construction and characterization of a modified strain (XcckatG), carrying an insertional mutation in the katG gene. First, we evaluated the involvement of KatG in the bacterial adaptive response to H₂O₂. XcckatG cultures exhibited lower catalase activity than those of the wild-type strain, and this activity was not induced upon treatment with sub-lethal doses of H₂O₂. Moreover, the KatG-deficient mutant exhibited decreased tolerance to H₂O₂ toxicity compared to wild-type cells and accumulated high intracellular levels of peroxides upon exposure to sub-lethal concentrations of H₂O₂. To further study the role of KatG in Xcc physiology, we evaluated bacterial survival upon exposure to UV-A or UV-B radiation. In both conditions, XcckatG showed a high mortality in comparison to Xcc wild-type. Finally, we studied the development of bacterial biofilms. While structured biofilms were observed for the Xcc wild-type, the development of these structures was impaired for XcckatG. Based on these results, we demonstrated that KatG is responsible for Xcc adaptive response to H₂O₂ and a key component of the bacterial response to oxidative stress. Moreover, this enzyme plays an important role during Xcc epiphytic survival, being essential for biofilm formation and UV resistance.     

The complete mitochondrial genome of Diaphorina citri (Hemiptera: Psyllidae) and phylogenetic analysis

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the most serious pest of citrus as the vector of Huanglongbing (HLB), the citrus greening disease. In this study, the complete mitochondrial genome (mitogenome) of D. citri has been sequenced and annotated, and a comparative analysis is provided with known Psylloidea species. The mitogenome of D. citri is a typical circular molecular of 15,038 bp in length with an A + T content of 74.56%, contains the typical 37 genes and the gene order is identical to the other Psylloidea mitogenomes. The nucleotide composition and codon usage of D. citri are similar to the four Psylloidea species. All protein-coding genes (PCGs) use standard initiation codons (TAN), stop with TAA and TAG except ND2 and ND5 which stop with incomplete termination codon T. All tRNAs have the typical clover-leaf structure, with the exception of trnS1 lacking the dihydrouridine (DHU) arm. The control region is located between rrnL and the trnI gene with the highest A + T content among the five Psylloidea species. Phylogenetic analysis is conducted based on the 13 PCGs or/and 2 rRNAs of 23 Sternorrhycha mitogenomes. Both maximum likelihood (ML) and Bayesian inference (BI) analysis suggest a clear relationship of Psylloidea, Aleyrodoidea and Aphidoidea within Sternorrhycha, which support the traditional morphological classification.     

A field study investigating the insecticidal efficacy against Diaphorina citri Kuwayama on Kinnow mandarin,Citrus reticulata Blanco trees

Asian citrus psyllid is a most damaging insect pest of citrus. In this field study, the efficacy of seven insecticides (emamectin benzoate, bifenthrin, chlorfenapyr, fipronil, imidacloprid, pyriproxyfen and thiamethoxam) was evaluated against Diaphorina citri Kuwayama in the citrus orchard of Kinnow mandarin, Citrus reticulata Blanco. The insecticides revealed a differential and substantial relative efficacy against D. citri compared to the untreated plants. The insecticidal effect attributed as percent reduction in insect population was more prominent after three days of spray: highest reduction values were recorded with thiamethoxam (50.89%), imidacloprid (44.27%) and bifenthrin (42.94%) after first spray, and thiamethoxam (83.36%), imidacloprid (73.20%) and bifenthrin (72.66%) after second spray. Thus, neonicotinoids (thiamethoxam and imidacloprid) and pyrethroid (bifenthrin) resulted as highly effective against D. citri at three days after both sprays. At seven days, imidacloprid (63.53%) and fipronil (62.47%) presented relatively higher population reduction after first spray, and thiamethoxam (92.66%) and chlorfenapyr (89.59%) after second spray. At 12 days, the insecticidal effect on insect population became significantly at par after each spray except chlorfenapyr that reflected high population reduction (93.17%) only after second spray. It is also obvious from the data that there is need of regular monitoring to suppress the psyllids population below threshold level by timely application of the second insecticidal spray.