GDP-mannose pyrophosphorylase is an efficient target in Xanthomonas citri for citrus canker control.

Xanthomonas citri subsp. citri (Xcc) is a bacterium that causes citrus canker, an economically important disease that results in premature fruit drop and reduced yield of fresh fruit. In this study, we demonstrated the involvement of XanB, an enzyme with phosphomannose isomerase (PMI) and guanosine diphosphate-mannose pyrophosphorylase (GMP) activities, in Xcc pathogenicity. Additionally, we found that XanB inhibitors protect the host against Xcc infection. Besides being deficient in motility, biofilm production, and ultraviolet resistance, the xanB deletion mutant was unable to cause disease, whereas xanB complementation restored wild-type phenotypes. XanB homology modeling allowed in silico virtual screening of inhibitors from databases, three of them being suitable in terms of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, which inhibited GMP (but not PMI) activity of the Xcc recombinant XanB protein in more than 50%. Inhibitors reduced citrus canker severity up to 95%, similarly to copper-based treatment. xanB is essential for Xcc pathogenicity, and XanB inhibitors can be used for the citrus canker control. IMPORTANCE: Xcc causes citrus canker, a threat to citrus production, which has been managed with copper, being required a more sustainable alternative for the disease control. XanB was previously found on the surface of Xcc, interacting with the host and displaying PMI and GMP activities. We demonstrated by xanB deletion and complementation that GMP activity plays a critical role in Xcc pathogenicity, particularly in biofilm formation. XanB homology modeling was performed, and in silico virtual screening led to carbohydrate-derived compounds able to inhibit XanB activity and reduce disease symptoms by 95%. XanB emerges as a promising target for drug design for control of citrus canker and other economically important diseases caused by Xanthomonas sp.

Xylose Isomerase Depletion Enhances Virulence of Xanthomonas citri subsp. citri in Citrus aurantifolia.

Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 (xylA1) and XAC4225 (xylA2), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes (xylA1 and/or xylA2) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence.

MALDI-TOF Mass Spectrometry for the Diagnosis of Citrus Canker Caused by Xanthomonas citri subsp. citri.

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), is a disease that causes serious problems to the global citrus industry. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has been used in human medicine to diagnose various diseases caused by both fungi and bacteria. In agriculture, this technique has potential for the diagnosis of diseases due to the low cost of large-scale analysis and quickness. This study showed that MALDI-TOF MS combined with chemometric analysis was effective for differentiating the macromolecule profile of orange leaves with canker lesions, healthy leaves, and leaves with phytotoxicity symptoms, proving that this technique may be used for the rapid diagnosis of citrus canker.

Late-Season Sweet Orange Selections Under Huanglongbing and Citrus Canker Endemic Conditions in the Brazilian Humid Subtropical Region.

The Brazilian citrus orchards are comprised by few genotypes, which increases the risk of pest and disease outbreaks. The diversification of sweet oranges (Citrus × sinensis) in orchards also generates off-season revenue and extend the fruit processing period. This study aimed to evaluate several horticultural traits of 19 late-season sweet orange selections under citrus canker and huanglongbing (HLB) endemic condition in northwestern Paraná state, Brazil, in a long-term field experiment. Tree size, yield, fruit quality for fresh fruit and industrial markets, estimates of tree density and yield, and citrus canker and huanglongbing (HLB) incidences were assessed. The experimental design was a randomized block with three replicates and five trees per unit. The orchard was drip-irrigated and arranged at tree spacing of 6.5 m × 4.5 m. All scions were graft-compatible with Rangpur lime (C. × limonia). Valencia selections had the tallest trees and largest canopies, particularly Olinda, Frost and #121 with heights and volumes greater than 4.20 m and 43 m3, respectively. Natal África do Sul and Whit's Late Valencia trees were the most productive with cumulative yields above 640 kg per tree. Most of the selections produced fruits of excellent physicochemical quality attending the fresh fruit and industrial market requirements. All selections showed similar horticultural characteristics for the fresh market, while Natal África do Sul and Charmute de Brotas were more suitable for juice processing. Frost Valencia and Valencia Late Fla. had the highest incidence of citrus canker on fruits (>20%), whereas IPR Folha Murcha, Charmute de Brotas and some Valencia selections (Chafeei Late, Campbell 479, Campbell 294, Olinda, Mutação and Whit's Late) exihibed low incidence (3.0-17.7%). At 9 years, Valencia Mutação trees had high HLB incidence (93%). In contrast, Natal IAC and Folha Murcha IAC showed the lowest HLB incidence (13%). Our results revealed that Natal IAC, Folha Murcha IAC, IPR Folha Murcha, Natal Murcha, Campbell 479 Valencia and Valencia Late Fla. had the best horticultural performance in addition to low HLB incidence. Together, these late-season sweet oranges are the most advantageous selections for citrus orchard diversification under citrus canker and HLB endemic conditions in humid subtropical regions.

Isolation and characterization of vB_XciM_LucasX, a new jumbo phage that infects Xanthomonas citri and Xanthomonas fuscans.

Citrus canker is one of the main bacterial diseases that affect citrus crops and is caused by Xanthomonas citri which affects all citrus species worldwide. New strategies to control citrus canker are necessary and the use of bacteriophages as biocontrol agent could be an alternative. Phages that infect Xanthomonas species have been studied, such as XacN1, a myovirus that infects X. citri. Here we report the isolation and characterization of a new jumbo phage, vb_XciM_LucasX, which infects X. citri and X. fuscans. Transmission electron microscopy allowed classification of LucasX in the Myoviridae family, which was corroborated by its genomic sequencing, annotation, and proteome clustering. LucasX has a 305,651 bp-long dsDNA genome. ORF prediction and annotation revealed 157 genes encoding putative structural proteins such as capsid and tail related proteins and phage assembly associated proteins, however, for most of the structural proteins it was not possible assign specific functions. Its genome encodes several proteins related to DNA replication and nucleotide metabolism, five putative RNA polymerases, at least one homing endonuclease mobile element, a terminase large subunit (TerL), an endolysin and many proteins classified as beneficial to the host. Proteome clustering and phylogeny analyses showed that LucasX is a new jumbo phage having as its closest neighbor the Xanthomonas jumbo phage Xoo-sp14. LucasX presented a burst size of 40 PFU/infected cell of X. citri 306, was completely inactivated at temperatures above 50°C, presented survival lower than 25% after 80 s of exposition to artificial UV light and had practically no tolerance to concentrations above 2.5 g/L NaCl or 40% ethanol. LucasX presented optimum pH at 7 and a broad range of Xanthomonas hosts, infecting twenty-one of the twenty-three strains tested. Finally, the LucasX yield was dependent on the host strain utilized, resulting one order of magnitude higher in X. fuscans C 752 than in X. citri 306, which points out to the possibility of phage yield improvement, an usual challenge for biocontrol purposes.

Impact of diseases and pests on premature fruit drop in sweet orange orchards in São Paulo state citrus belt, Brazil.

BACKGROUND: Despite technical improvements in the citrus chain and leadership in orange production achieved in the past decades, premature fruit drop remains a major component of crop loss in São Paulo state citrus belt, the largest sweet orange production area in the world. The present study aimed to determine, during five consecutive seasons, the impact of the diseases and pests on premature fruit drop in the orange belt. RESULTS: Fruit drop due to the main diseases and pests averaged approximately 11.0%, which corresponded to approximately 63% of the annual fruit drop. The average fruit drop rate due to fruit borer and fruit flies combined was 4.0%, Huanglongbing (HLB) 3.3%, black spot 2.6%, leprosis 1.0% and citrus canker 0.3%. The average amount of fruit drop (million 40.8 kg boxes) and value of crop losses (million US$ dollars), in five seasons, were 12.7 and 66.2 for fruit borer/fruit flies, 11.0 and 57.9 for HLB, 8.1 and 42.2 for black spot, 3.1 and 15.6 for leprosis, and 0.9 and 4.9 for citrus canker, respectively. CONCLUSION: Fruit borer and fruit flies (combined), HLB, black spot, leprosis and citrus canker are, in this order, the main diseases and pests in the orange belt of São Paulo state. All of these causes significantly increased the overall fruit drop rate in the evaluated seasons. The results will contribute to the development of the Brazilian citrus industry, while showing to other citrus-growing regions the potential that diseases and pests have to jeopardize production. © 2022 Society of Chemical Industry.

Tree age and cultivar-oriented use of mineral oil added to fungicide tank mixture for the control of citrus black spot in sweet orange orchards.

BACKGROUND: Mineral oil added to fungicide spray mixtures has been a frequently used strategy to control citrus black spot (CBS) worldwide. Although mineral oil may increase the efficacy of control, its use represents around 15% of the costs of a CBS spray program. This study aimed to assess the performance of different proportions of mineral oil added to a fungicide tank mixture for CBS control in young (less than 10 years old) and old (more than 12 years old) sweet orange orchards of early ('Hamlin'), mid-season ('Pera') and late-maturing ('Valencia') cultivars in São Paulo state, Brazil. The efficacy of 0.15%, 0.20% or 0.25% mineral oil added to a fungicide spray mixture was determined by assessing CBS incidence, severity and fruit drop in six orchards over two seasons. RESULTS: Fungicide programs with or without oil were effective in reducing 100% CBS symptom expression in both young and old 'Hamlin' orchards and in the young 'Pera' orchard. The lowest mineral oil rate tested (0.15%) showed a reduction in CBS intensity of around 90%, similar to the highest rate tested in the old 'Pera' orchard. The highest cost-benefit program to control CBS in the old 'Valencia' orchard was obtained with the mineral oil rate of 0.25%, commonly used in the São Paulo citrus belt, which reduced CBS severity by up to 97%. CONCLUSION: This study demonstrated that mineral oil rates for CBS control can be adjusted according to tree age and cultivar. These findings contribute to the establishment of more sustainable citrus production by reducing spray costs while maintaining the efficacy of CBS control. © 2021 Society of Chemical Industry.

Relative Contribution of Windbreak, Copper Sprays, and Leafminer Control for Citrus Canker Management and Prevention of Crop Loss in Sweet Orange Trees.

The management of citrus canker, caused by Xanthomonas citri subsp. citri, has been widely studied in endemic areas because of the importance of the disease in several citrus-producing countries. A set of control measures is well established, but no study has investigated the efficiency of each measure individually and their combination for disease suppression. This study comprised a 3-year field study to assess the relative contribution of three measures for the control of citrus canker and reduction of crop losses. Windbreak (Wb), copper sprays (Cu), and leafminer control (Lc) were assessed in eight different combinations in a split-split plot design. The orchard was composed of 'Valencia' sweet orange trees grafted onto 'Rangpur' lime. Casuarina cunninghamiana trees were used as Wb. Cu and Lc sprays were performed every 21 days throughout the year. Individually, Cu showed the highest contribution for canker control, followed by Wb. Lc had no effect on reducing citrus canker. Wb+Cu showed the highest efficiency for control of the disease. This combination reduced the incidence of diseased trees by approximately 60%, and the incidence of diseased leaves and fruit by ≥90% and increased the yield in 2.0- to 2.6-fold in comparison with the unmanaged plots. Cu sprays were important for reducing disease incidence and crop losses, whereas Wb had an additional contribution in minimizing the incidence of cankered, non-marketable fruit. The results indicated that the adoption of these measures of control may depend on the characteristics of the orchard and destination of the production.

Spray Volume and Rate Based on the Tree Row Volume for a Sustainable Use of Copper in the Control of Citrus Canker.

Copper is the most efficient pesticide for the control of citrus canker (Xanthomonas citri subsp. citri). To mitigate the environmental impacts and costs, the copper sprays in citrus orchards are being optimized based on the tree row volume (TRV). A previous investigation allowed for significant reductions of the spray volume and copper rates. Nevertheless, the results also indicated the need for additional studies. The aim of this work was to assess whether both the spray volume and the metallic copper rate based on the TRV may be further reduced. A field trial was carried out during two seasons in a 3-year-old commercial orchard of Pera sweet orange located in the municipality of Paranavaí, Paraná, Brazil. The volumes of 20 and 40 ml of spray mixture per m3 of the tree canopy were assessed in combination with the metallic copper rates of 10.5, 21.0, 36.8, or 52.5 mg/m3. Disease was measured as the temporal progress of canker incidence on leaves, cumulative dropped fruit with canker, and incidence of diseased fruit at harvest. The quality of sprays was assessed by measuring the copper deposition and leaf coverage. The treatment with the highest citrus canker control for the lowest use of water and copper was the combination of 40 ml and 36.8 mg/m3. Regression analyses indicated that the minimum threshold deposition of copper was ∼1.5 µg Cu2+/cm2 leaf area. In addition, the lowest spray volume and copper rate necessary to achieve this deposition are 35 ml/m3 and 30 mg/m3. The use of 20 ml/m3 did not efficiently control the disease due to the deficient coverage of treated surfaces. This study demonstrated that it is possible to use even lower amounts of copper and water without interfering with the efficiency of control of citrus canker.

Hexyl gallate for the control of citrus canker caused by Xanthomonas citri subsp citri.

Brazil is the biggest producer of sweet oranges and the main exporter of concentrated orange juice in the world. Among the diseases that affect citriculture, Asiatic citrus canker, caused by the bacterial pathogen Xanthomonas citri, represents one of the most significant threats. The current Brazilian legislation regulating the control of citrus canker no longer requires the eradication of affected trees in states where the incidence of the disease is high. Instead, control involves disease control measures, including periodic preventative spraying of copper compounds. The long-term use of copper for plant disease control has raised concerns about environmental accumulation and toxicity, as well as the selective pressure it exerts leading to the emergence of copper-resistant X. citri strains. Here, we evaluated hexyl gallate (G6) as an alternative to copper compounds for citrus plant protection. G6 was able to protect citrus nursery trees against X. citri infection. Thirty days after inoculation, the trees treated with G6 developed 0.5 lesions/cm2 leaf area compared with the 2.84 lesions/cm2 observed in the untreated control trees. Also, G6 did not interfere with germination and root development of tomato, lettuce, and arugula, which is consistent with our previous data showing that G6 is safe for tissue culture cell lines. Membrane permeability tests showed that the primary target of G6 is the bacterial outer membrane. Finally, we could not isolate spontaneous X. citri mutants resistant to G6 nor induce resistance to G6 after long-term exposures to increasing concentrations of the compound, which suggests that G6 may have multiple cellular targets. This study demonstrated that G6 is a promising candidate for the development and use in citrus canker management.

Characteristics of Citrus Canker Lesions Associated with Premature Drop of Sweet Orange Fruit.

During the development of a citrus fruit, many cycles of infection by Xanthomonas citri subsp. citri may occur leading to the development of a range of characteristics of citrus canker lesions scattered across the fruit surface. This study aimed to determine whether the size of the lesions, their distance from the peduncle, and the number and time of appearance of the lesions on fruit of sweet orange were associated with premature fruit drop. A multiple linear regression analysis revealed a negative relationship between the fruit detachment force and the lesion diameter, the proximity of the nearest lesion to the peduncle and the number of lesions. A survival analysis demonstrated that these characteristics significantly influenced the probability and the time that a cankered fruit remained attached to the tree. More than 90% of dropped fruit had large lesions (>5 mm) but not all fruit with large lesions dropped before harvest. Approximately 50% of the harvested fruit had lesions >5 mm. On the harvested fruit remaining on the tree, although large, the lesions had a smaller diameter, were located farther from the peduncle, and were less numerous than those observed on dropped fruit. Small canker lesions neither reduced the detachment force nor the survival of fruit in the tree. The earlier a fruit expressed canker symptoms, the higher the probability the fruit developed large lesions near the peduncle and/or developed lesions in greater numbers. This study provides a better understanding on the relationship between the time of appearance of lesions of citrus canker on fruit and premature fruit drop. This information defines the critical period for fruit protection and may be used to improve disease management.

Bioguided isolation, characterization and media optimization for production of Lysolipins by actinomycete as antimicrobial compound against Xanthomonas citri subsp. citri.

Citrus Canker disease is one of the most important disease in citrus production worldwide caused by gram-negative bacterial pathogen Xanthomonas citri subsp. citri, leading to great economic losses. Currently, a spray of copper-based bactericides is the primary measure for citrus canker management. However, these measures can lead to the contamination of soil by metal contamination, but also the development of copper-resistant Xanthomonas populations. Considering the need to discovery new alternatives to control the citrus canker disease, actinomycetes isolated from the Brazilian Caatinga biome and their crude extracts were tested against different strains of Xanthomonas citri subsp. citri. Streptomyces sp. Caat 1-54 crude extract showed the highest antibiotic activity against Xcc. The crude extract dereplication was performed by LC-MS/MS. Through bioassay-guided fractionation strategy, the antimicrobial activity was assigned to Lysolipins, showing a MIC around 0.4-0.8 µg/mL. Growth media optimization using statistical experimental design increased the Lysolipins production in three-fold production. The preventive and curative effects of the optimized crude extract obtained by experimental design of Caat-1-54 against citrus canker were evaluated in potted 'Pera' sweet orange nursery trees. Caat 1-54 extract was effective in preventing new infections by Xcc on leaves but was not able to reduce Xcc population in pre-established citrus canker lesions. Streptomyces sp. Caat 1-54 extract is a promising, environmentally-friendly source of antimicrobial compound to protect citrus trees against citrus canker.

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.

Critical Fungicide Spray Period for Citrus Black Spot Control in São Paulo State, Brazil.

The period of citrus black spot (CBS) control used in South Africa (SA) and Australia, from October to January or February, has not been as effective in São Paulo (SP), Brazil. This study aimed to evaluate different periods of protection and determine the critical period for CBS control in SP. A field trial was carried out for two seasons in a mature Valencia sweet orange orchard located in Mogi Guaçu, SP. Spray programs with a total of 60, 100, 140, 180, and 220 days of fruit protection (DFP) were evaluated. CBS symptoms and fruit drop decreased exponentially as the length of the period of protection increased. The reductions in CBS intensity and crop loss with these programs ranged from 34 to 96 and 50 to 77%, respectively. The programs with 180 and 220 DFP, which protected the fruit from September to March and May, showed the highest cost benefit. The critical period needed for CBS control in SP is longer than that in SA and Australia. The results obtained with the present study are helpful for scheduling a more efficient and rational program for CBS control not only in SP but also in other tropical and subtropical regions with similar weather conditions.

Evidence for acquisition of copper resistance genes from different sources in citrus-associated xanthomonads.

ABSTRACT We determined that multiple and independent introductions of copper resistance genes have taken place for strains of Xanthomonas citri subsp. citri from Argentina and strains of X. alfalfae subsp. citrumelonis from Florida. This study compared the partial nucleotide sequences of principal copper resistance genes copL, copA, and copB from X. citri subsp. citri and X. alfalfae subsp. citrumelonis to strains of other Xanthomonas spp. resistant to copper that were isolated from 12 different countries or territories. The survey confirmed that the copLAB gene cluster is present in many species of Xanthomonas from different parts of the world. Alignment of partial nucleotide sequences of copper resistance genes among the copper-resistant (Cu(R)) strains of Xanthomonas detected homology of ≥92, ≥96, and ≥91% for copL, copA, and copB, respectively. Grouping of strains based on branching patterns of phylogenetic trees was similar for copL and copA but differed for copB. When the three genes were concatenated and analyzed using various phylogenetic methods, it appeared that the plasmid had been horizontally transferred and various populations were mutating based on selection pressure unique to geographic regions. Although high homology of the genes among the strains indicated that the copper resistance in xanthomonads has a common origin, the slight differences in nucleotide sequences within groups of strains indicated that Cu(R) genes have been independently exchanged among species of Xanthomonas throughout the world.

Determinação da capacidade de uso do solo visando o manejo sustentável para uma média propriedade em Londrina-PR / Determination of use of land aimed for sustainable management on medium size farm in Londrina-PR

O planejamento conservacionista remete ao estabelecimento de um esquema de trabalho para a propriedade agrícola, de tal forma que assegure a conservação do solo juntamente com sua exploração lucrativa. A capacidade do uso das terras indica o grau de cultivo que se pode aplicar em um terreno sem que o solo sofra diminuição da sua produtividade por efeito da erosão. Desta forma, o objetivo do trabalho foi realizar o levantamento do meio físico no ano de 2010 e classificação das terras no sistema de capacidade de uso para uma média propriedade brasileira, localizada no município de Londrina (PR) sobre Latossolo Vermelho e Nitossolo Vermelho para aprimorar a exploração conservacionista da área com a introdução da taxa de adoção de práticas conservacionistas. O mapeamento da área foi executado por meio de um aparelho portátil de georeferenciamento. Baseado nos dados levantados, foram identificadas diversas classes de capacidade de uso do solo: II (culturas anuais), III (perenes ou anuais), IV (perenes ou pastagem), V (pastagem) e VII (reflorestamento). Assim, com o conhecimento das classes de capacidade de uso do solo da propriedade foi possível realizar um planejamento voltado ao manejo conservacionista visando a correta utilização do solo e determinar o pagamento de taxa de adoção de práticas conservacionistas, para a destinação das glebas 2 e 3 (Cambissolo) para a implantação de Reserva Legal.

The conservation planning of a farm refers to the sustainable use of the land. The bearing capacity of a soil indicates to what extent the land can be exploited without mining the natural resources and original characteristics due to erosion. The objective of this work was to assess the soil attributes of a medium size brazilian farm in 2010, located in Londrina, Paraná, Brazil, to optimize land conservationist use with the introduction of the rate of adoption of conservation practices. The area was mapped using a geographical position system (GPS) device and the soil was analyzed chemically and visually. Five classes of soil were identified in the property: II (annual crops), III (perennial or annual crops), IV (perennial crops or pasture), V (pasture) and VII (reforestation). The classification of the soil allowed the establishment of a conservation plan, which aims at maximizing the use to the land without impacting the environment. side, the payment of rate of adoption of conservation practices, to destination of areas 2 and 3 (Cambisols, Inceptisols) for the implantation of Legal Reserve.

Effect of Application Frequency and Reduced Rates of Acibenzolar-S-Methyl on the Field Efficacy of Induced Resistance Against Bacterial Spot on Tomato.

Acibenzolar-S-methyl (ASM), a plant activator known to induce systemic acquired resistance, has demonstrated an ability to manage a number of plant diseases, including bacterial spot on tomato caused by four distinct Xanthomonas spp. The aim of this study was to evaluate application rate and frequency of ASM in order to optimize field efficacy against bacterial spot in Florida, while minimizing its impact on marketable yields. ASM was applied biweekly (once every 2 weeks) as a foliar spray at a constant concentration of 12.9, 64.5, and 129 µM throughout four field experiments during 2007-08. A standard copper program and an untreated control were also included. Overall, biweekly applications of ASM did not significantly reduce disease development or the final disease severity of bacterial spot compared with the copper-mancozeb standard or the untreated control. Only one experiment showed a significant reduction in the final disease severity on plants treated with ASM at 129 µM compared with the untreated control. Three additional field trials conducted during 2009-10 to evaluate the effects of weekly and biweekly applications of ASM at concentrations of 30.3 to 200 µM found that weekly applications provided significantly better disease control than biweekly applications. The tomato yields were not statistically improved with the use of ASM relative to the untreated control and standard copper program. Weekly ASM applications at rates as low as 75 µM (equivalent to 1.58 g a.i./ha in 100 liters of water or 0.21 oz. a.i./acre in 100 gallons of water) to 200 µM (equivalent to 4.20 g a.i./ha in 100 liters of water or 0.56 oz. a.i./acre in 100 gallons of water) were statistically equivalent in managing bacterial spot of tomato without significantly reducing yield compared with the untreated control.

Molecular characterization of copper resistance genes from Xanthomonas citri subsp. citri and Xanthomonas alfalfae subsp. citrumelonis.

Copper sprays have been widely used for control of endemic citrus canker caused by Xanthomonas citri subsp. citri in citrus-growing areas for more than 2 decades. Xanthomonas alfalfae subsp. citrumelonis populations were also exposed to frequent sprays of copper for several years as a protective measure against citrus bacterial spot (CBS) in Florida citrus nurseries. Long-term use of these bactericides has led to the development of copper-resistant (Cu(r)) strains in both X. citri subsp. citri and X. alfalfae subsp. citrumelonis, resulting in a reduction of disease control. The objectives of this study were to characterize for the first time the genetics of copper resistance in X. citri subsp. citri and X. alfalfae subsp. citrumelonis and to compare these organisms to other Cu(r) bacteria. Copper resistance determinants from X. citri subsp. citri strain A44(pXccCu2) from Argentina and X. alfalfae subsp. citrumelonis strain 1381(pXacCu2) from Florida were cloned and sequenced. Open reading frames (ORFs) related to the genes copL, copA, copB, copM, copG, copC, copD, and copF were identified in X. citri subsp. citri A44. The same ORFs, except copC and copD, were also present in X. alfalfae subsp. citrumelonis 1381. Transposon mutagenesis of the cloned copper resistance determinants in pXccCu2 revealed that copper resistance in X. citri subsp. citri strain A44 is mostly due to copL, copA, and copB, which are the genes in the cloned cluster with the highest nucleotide homology (≥ 92%) among different Cu(r) bacteria.

Comparative genomics reveals diversity among xanthomonads infecting tomato and pepper.

BACKGROUND: Bacterial spot of tomato and pepper is caused by four Xanthomonas species and is a major plant disease in warm humid climates. The four species are distinct from each other based on physiological and molecular characteristics. The genome sequence of strain 85-10, a member of one of the species, Xanthomonas euvesicatoria (Xcv) has been previously reported. To determine the relationship of the four species at the genome level and to investigate the molecular basis of their virulence and differing host ranges, draft genomic sequences of members of the other three species were determined and compared to strain 85-10. RESULTS: We sequenced the genomes of X. vesicatoria (Xv) strain 1111 (ATCC 35937), X. perforans (Xp) strain 91-118 and X. gardneri (Xg) strain 101 (ATCC 19865). The genomes were compared with each other and with the previously sequenced Xcv strain 85-10. In addition, the molecular features were predicted that may be required for pathogenicity including the type III secretion apparatus, type III effectors, other secretion systems, quorum sensing systems, adhesins, extracellular polysaccharide, and lipopolysaccharide determinants. Several novel type III effectors from Xg strain 101 and Xv strain 1111 genomes were computationally identified and their translocation was validated using a reporter gene assay. A homolog to Ax21, the elicitor of XA21-mediated resistance in rice, and a functional Ax21 sulfation system were identified in Xcv. Genes encoding proteins with functions mediated by type II and type IV secretion systems have also been compared, including enzymes involved in cell wall deconstruction, as contributors to pathogenicity. CONCLUSIONS: Comparative genomic analyses revealed considerable diversity among bacterial spot pathogens, providing new insights into differences and similarities that may explain the diverse nature of these strains. Genes specific to pepper pathogens, such as the O-antigen of the lipopolysaccharide cluster, and genes unique to individual strains, such as novel type III effectors and bacteriocin genes, have been identified providing new clues for our understanding of pathogen virulence, aggressiveness, and host preference. These analyses will aid in efforts towards breeding for broad and durable resistance in economically important tomato and pepper cultivars.