Control Of Canker Research Articles

Bactericidal Effect of Entomopathogenic Bacterium Pseudomonas entomophila Against Xanthomonas citri Reduces Citrus Canker Disease Severity.

The bacterium Pseudomonas entomophila has been recognized as an exceptional species within the Pseudomonas genus, capable of naturally infecting and killing insects from at least three different orders. P. entomophila ingestion leads to irreversible gut damage resulting from a global blockage of translation, which impairs both immune and tissue repair systems in the insect intestine. In this study we isolated a P. entomophila bacterial strain from soil samples which displayed a strong activity against Xanthomonas citri subsp, citri (Xcc), the etiological agent of citrus canker disease. The antagonism potential of isolated bacteria against Xcc and its ability to reduce citrus canker severity was assessed both ex planta and in planta. Our findings show that pathogenicity assays in Citrus x limonia by pressure infiltration and spray with a mixture of P. entomophila and Xcc leaded to a significant reduction in the number of canker lesions in high susceptible citrus leaves, at 21 days post-infection. To the best of our knowledge this is the first report of antibacterial activity of P. entomophila against a phytopathogenic bacterium. Collective action of P. entomophila factors such as diketopiperazine production and the type 6 secretion system (T6SS) may be involved in this type of biological control of citrus canker. The results suggest that the P. entomophila strain could be a promising biocontrol agent acting directly against Xcc.

Xanthomonas citri subsp. citri: host interaction and control strategies

Citrus is one of the most ancient fruit crops cultivated in the world. For many countries, citrus production is an important source of revenues. However, despite the richness of citrus production worldwide, fruit yields are constantly threatened by diseases that cause serious economic and social impacts to growers and consumers. One such example is citrus canker, a disease that affects all commercial citrus varieties and for which there is no cure. Currently, control measures for citrus canker are restricted to the application of copper-based compounds and the elimination of infected trees to prevent pathogen spreading in the field. However, new alternatives for the control of this disease are being developed, spanning through transgenic plants to innovative chemicals and biological control. New genome editing approaches and a repertoire of plant defense-related genes have been exploited to produce citrus lineages more tolerant to X. citri. In addition, a series of new molecules have been tested to not only inhibit X. citri growth but to also boost the plant immune system to suppress disease progression. In this review, we provide the state of art of all citrus canker control measures in use today, highlighting their utility and drawbacks and commenting on novel strategies to better control this important citrus disease.

Development of genetically modified citrus plants for the control of citrus canker and huanglongbing

Citrus cultivation is challenging due to the plethora of abiotic and biotic stresses faced by the crop. In recent years, production has been severely affected by diseases such as citrus canker and huanglongbing (HLB). Disease management is hampered as there is no field resistance to these diseases in any of the important commercially planted varieties. Traditionally, conventional breeding approaches have been applied for the improvement of the susceptible cultivars; however, this technique is laborious and time consuming. Genetic transformation of citrus allows for the rapid integration of novel genes into the plant’s genome to develop disease-resistant transgenic plants. Therefore, efforts have been made to utilize genetic engineering tools to develop genetically modified citrus that are resistant to citrus canker and HLB. This review summarizes the major achievements made in the development of citrus canker and HLB tolerance using transgenic technologies.

In vitro antimicrobial activity of plant extracts against Pseudomonas syringae pv. actinidiae causal agent of bacterial canker in kiwifruit

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker of kiwifruit, is considered the main pathogen of yellow-, green- and red-fleshed kiwifruit. All major kiwifruit producing countries in the world have been affected by this bacterial pathogen, leading to substantial economic losses. The control of bacterial canker of kiwifruit is based only on preventive methods or on the use of copper compounds that can cause phytotoxicity problems. In this study, the in vitro antibacterial activity of seven different plant extracts against eight Psa strains has been evaluated. The inhibition of 100% of the Psa growth was observed, after 24 h, for the extracts of Polygonum cuspidatum roots (POL-roots), Hypericum perforatum roots elicited with chitosan oligosaccharides (HYP-COS roots) and non-fermented grape pomace (ITA-pomace). The strongest antibacterial activity was exhibited by POL-roots, with a geometric mean of minimum inhibitory concentration of 100% of growth (GMMIC100) of 105.11 µg/mL after 24 h, and with a GMMIC100 value of 148.65 µg/mL after 48 h. Moreover, POL-roots extract showed the best bactericidal activity with a GMMBC of 210.22 µg/mL. No phytotoxic activity was observed up to 15 days in the leaves of Actinidia chinensis “Belen” treated with plant extracts at 500 µg/mL.

Control of Citrus Canker in Greenhouse and Field with a Zinc, Urea, and Peroxide Ternary Solution.

Accumulation of toxic copper in soil and development of copper-resistant pests are emerging challenges currently faced by the agricultural community worldwide. As an alternative, we have developed a ternary zinc chelate solution (TSOL) pesticide where zinc ions are the primary active ingredient. The material is composed of zinc, urea, and hydrogen peroxide. Urea was chosen as it is widely used as a plant fertilizer and can also bind to both zinc and hydrogen peroxide. No phytotoxicity was observed with TSOL on Meyer lemon (Citrus × meyeri) seedlings at a field spray rate of 800 μg/mL Zn metal concentration. Antimicrobial studies showed that TSOL exhibited improved killing efficacy against Escherichia coli and Xanthomonas alfalfae compared to Zn ions alone. Citrus canker field trials in a grapefruit (Chrysopelea paradisi) grove over three years showed that TSOL provided comparable disease protection to copper products at an equivalent or lower metal content.

Evaluation of Various Chemicals Against Citrus Canker on Grapefruit cv. Shamber

A good quality fruit fetches a high market value. Citrus canker disease badly affects quality of citrus fruits including grapefruit. The present study was conducted to find out suitable chemicals, alone or incombination, for the control of citrus canker. The experiment was conducted on 15-year-old disease-affected grapefruit cv. Shamber plants in the orchard of Horticultural Research Station, Sahiwal to control the disease for better quality fruit production. Therefore, four sprays of Aliette (300 g 100 L-1 of water), Bordeaux mixture (1:1:100), Flare (100 g 100 L-1 of water) or Bordeaux mixture + Flare were applied; two in the month of March and other two in the month of August with fifteen days interval along with control (no chemical). Combined application of Bordeaux mixture and Flare reduced the attack of citrus canker on leaves and fruits. Comparatively lower values of affected leaves (1.27%), lesions per leaf (0.40), affected fruits (0.25 %) and lesions per fruit (0.22) were observed in the treatment in which a combination of Bordeaux mixture and Flare was sprayed on the plants. The disease percentage was high on leaves and fruits of unsprayed (control) plants, while other thee treatment were in the middle in their efficacy to control the disease.

Foliar Application of Limewash Improves Vegetative and Reproductive Growth and Fruit Quality of Bell Pepper under Deficit Irrigation

A good quality fruit fetches a high market value. Citrus canker disease badly affects quality of citrus fruits including grapefruit. The present study was conducted to find out suitable chemicals, alone or incombination, for the control of citrus canker. The experiment was conducted on 15-year-old disease-affected grapefruit cv. Shamber plants in the orchard of Horticultural Research Station, Sahiwal to control the disease for better quality fruit production. Therefore, four sprays of Aliette (300 g 100 L-1 of water), Bordeaux mixture (1:1:100), Flare (100 g 100 L-1 of water) or Bordeaux mixture + Flare were applied; two in the month of March and other two in the month of August with fifteen days interval along with control (no chemical). Combined application of Bordeaux mixture and Flare reduced the attack of citrus canker on leaves and fruits. Comparatively lower values of affected leaves (1.27%), lesions per leaf (0.40), affected fruits (0.25%) and lesions per fruit (0.22) were observed in the treatment in which a combination of Bordeaux mixture and Flare was sprayed on the plants. The disease percentage was high on leaves and fruits of unsprayed (control) plants, while other thee treatment were in the middle in their efficacy to control the disease.

N-Acyl Homoserine Lactones and Lux Solos Regulate Social Behaviour and Virulence of Pseudomonas syringae pv. actinidiae.

The phyllosphere is a complex environment where microbes communicate through signalling molecules in a system, generally known as quorum sensing (QS). One of the most common QS systems in Gram-negative proteobacteria is based on the production of N-acyl homoserine lactones (AHLs) by a LuxI synthase and their perception by a LuxR sensor. Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit, colonises plant phyllosphere before penetrating via wounds and natural openings. Since Psa genome encodes three LuxR solos without a cognate LuxI, this bacterium may perceive diffusible signals, but it cannot produce AHLs, displaying a non-canonical QS system. The elucidation of the mechanisms underlying the perception of environmental cues in the phyllosphere by this pathogen and their influence on the onset of pathogenesis are of crucial importance for a long-lasting and sustainable management of the bacterial canker of kiwifruit. Here, we report the ability of Psa to sense its own population density and the presence of surrounding bacteria. Moreover, we show that Psa can perceive AHLs, indicating that AHL-producing neighbouring bacteria may regulate Psa virulence in the host. Our results suggest that the ecological environment is important in determining Psa fitness and pathogenic potential. This opens new perspectives in the use of more advanced biochemical and microbiological tools for the control of bacterial canker of kiwifruit.

Description of copper tolerantXanthomonas citrisubsp.citriand genotypic comparison with sensitive and resistant strains

The copper‐based products widely used for control of citrus canker may lead to the development ofXanthomonas citrisubsp.citri(X. citri) resistant to copper (CuR). However, the study of copper sensitivity ofX. citristrains from Paraná state, Brazil, did not reveal the existence of CuR, but copper tolerant (CuT) strains. The aim of this study was to describe for the first time the existence of CuTX. citriand compare the genetic determinants that differentiate the CuTstrains from the sensitive (CuS) and CuRstrains. CuTstrains supported intermediate concentrations of copper in comparison to CuSand CuR. CuTstrains lack the gene clusterscopLABorcopABCDresponsible for copper resistance in CuRstrains and the large plasmids (c. ≥200 kb) that normally carry these genes. The nucleotide sequences of chromosomal homologous genescohLAB, involved in copper homeostasis, were 100% similar in strains of all phenotypes. CuTstrains differed from CuSstrains by the higher expression of the homologous chromosomal genescohAandcohBin the presence of copper. CuTX. citristrains are not precursors of CuRstrains and do not pose a threat to the efficient use of copper‐based bactericides for management of citrus canker in citrus orchards. Copper resistance and tolerance are distinct phenotypes and should not be used as synonyms. The proper characterization of the sensitivity to copper leads to a more confident monitoring of the distribution of copper resistant populations ofX. citriand adoption of containment measures only when necessary.

Efficacy of Fungicide Treatments for Control of Anthracnose Canker in Young Cider Apple Trees in Western Washington

Anthracnose canker, caused by Neofabraea malicorticis, threatens the sustainability of cider apple (Malus ×domestica) production in the maritime climate of western Washington. In the short-term, the disease reduces overall orchard productivity and in the long-term it reduces an orchard’s economic life span. The disease is difficult to manage using cultural practices, and information on fungicide efficacy is limited and contradictory. To address this situation, a 2-year study was conducted to evaluate efficacy of zinc (4.49 lb/acre), basic copper sulfate (2.49 lb/acre), captan (2.94 lb/acre), thiophanate-methyl (0.69 lb/acre), pyraclostrobin plus boscalid (0.38 lb/acre), and combinations of these fungicides to manage anthracnose canker infection in young cider apple trees cultivated in a maritime climate. Trees used in the first year of the study (2016) were found to be infected by anthracnose canker on receipt, so the first year was a measure of disease control and the second year (2017) was a measure of disease prevention. In 2016, when fungicide treatments were applied every 3 weeks from March through October, none of the treatments evaluated inhibited the development of new infections or the expansion of existing cankers (77% increase in canker size on average for all treatments). In 2017, when fungicide treatments were applied every 3 weeks from February through April, two to three new cankers were observed 3 weeks after final treatment application for all treatments. Results from this study demonstrate that the current fungicides recommended for control of anthracnose canker are not reliably effective in the orchard environment of northwest Washington. Future studies should assess the fungicides evaluated in this study applied in rotation with additional systemic fungicides.

Novel Plastid-Nuclear Genome Combinations Enhance Resistance to Citrus Canker in Cybrid Grapefruit.

Host disease resistance is the most desirable strategy for control of citrus canker, a disease caused by a gram-negative bacterium Xanthomonas citri subsp. citri. However, no resistant commercial citrus cultivar has been identified. Cybridization, a somatic hybridization approach that combines the organelle and nuclear genomes from different species, was used to create cybrids between citrus canker resistant ‘Meiwa’ kumquat (Fortunella crassifolia Swingle snym. Citrus japonica Thunb.) and susceptible grapefruit (Citrus paradisi Macfad) cultivars. From these fusions, cybrids with grapefruit nucleus, kumquat mitochondria and kumquat chloroplasts and cybrids with grapefruit nucleus, kumquat mitochondria and grapefruit chloroplasts were generated. These cybrids showed a range of citrus canker response, but all cybrids with kumquat chloroplasts had a significantly lower number of lesions and lower Xanthomonas citri subsp. citri populations than the grapefruit controls. Cybrids with grapefruit chloroplasts had a significantly higher number of lesions than those with kumquat chloroplasts. To understand the role of chloroplasts in the cybrid disease defense, quantitative PCR was performed on both cybrid types and their parents to examine changes in gene expression during Xanthomonas citri subsp. citri infection. The results revealed chloroplast influences on nuclear gene expression, since isonuclear cybrids and ‘Marsh’ grapefruit had different gene expression profiles. In addition, only genotypes with kumquat chloroplasts showed an early up-regulation of reactive oxygen species genes upon Xanthomonas citri subsp. citri infection. These cybrids have the potential to enhance citrus canker resistance in commercial grapefruit orchards. They also serve as models for understanding the contribution of chloroplasts to plant disease response and raise the question of whether other alien chloroplast genotypes would condition similar results.

Endophytic Bacteria Isolated from Citrus Plants for Biological Control of Citrus Canker in Lime Plants.

Citrus canker caused by Xanthomonas citri subsp. citri is a disease affecting the yield and fruit quality of lime (Citrus aurantiifolia). This research investigated endophytic bacteria obtained from six healthy Citrus spp. to inhibit the pathogen and to control citrus canker on lime plants. Numbers of the endophytic bacteria isolated from C. aurantifolia, C. hystrix, C. maxima, C. nobilis, C. reticulata and C. sinensis were 28, 25, 29, 42, 12 and 34 isolates, respectively. The selected endophytic bacteria that were effective against X. citri subsp. citri were Bacillus amyloliquefaciens LE109, B. subtilis LE24 and B. tequilensis PO80. The optimum culture medium for an antagonistic effect on the pathogen in B. amyloliquefaciens LE109 and B. tequilensis PO80 was yeast extract peptone dextrose broth, and in B. subtilis LE24 was modified soluble starch broth. To control citrus canker in lime, young expanded leaves of lime plants were aseptically punctured and inoculated with 30 μl of bacterial suspension of the pathogen (108 CFU/ml in 0.85% NaCl) per punctured location. After the pathogenic inoculation for 24 h, the leaves were then inoculated with 30 μl of the selected endophytic bacteria (108 CFU/ml in 0.85% NaCl), and treated with 30 μl of the culture media containing bioactive compounds produced by the selected endophytic bacteria. The leaves inoculated with cell suspensions of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. However, the leaves inoculated with B. tequilensis PO80 displayed 10% disease incidence. Additionally, the leaves treated with the crude bioactive compounds of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. Notably, the leaves treated with the crude bioactive compounds of B. tequilensis PO80 displayed 5% disease incidence. The results of this study showed that the Bacillus strains play important roles in the biocontrol of citrus canker in lime.

Design of Antibacterial Agents: Alkyl Dihydroxybenzoates against Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) causes citrus canker, affecting sweet orange-producing areas around the world. The current chemical treatment available for this disease is based on cupric compounds. For this reason, the objective of this study was to design antibacterial agents. In order to do this, we analyzed the anti-Xcc activity of 36 alkyl dihydroxybenzoates and we found 14 active compounds. Among them, three esters with the lowest minimum inhibitory concentration values were selected; compounds 4 (52 μM), 16 (80 μM) and 28 (88 μM). Our study demonstrated that alkyl dihydroxybenzoates cause a delay in the exponential phase. The permeability capacity of alkyl dihydroxybenzoates in a quarter of MIC was compared to nisin (positive control). Compound 28 was the most effective (93.8), compared to compound 16 (41.3) and compound 4 (13.9) by percentage values. Finally, all three compounds showed inhibition of FtsZ GTPase activity, and promoted changes in protofilaments, leading to depolymerization, which prevents bacterial cell division. In conclusion, heptyl dihydroxybenzoates (compounds 4, 16 and 28) are promising anti-Xcc agents which may serve as an alternative for the control of citrus canker.

Purification and structural characterization of fengycin homologues produced by Bacillus subtilis from poplar wood bark

Poplar fungi canker is a disease that constitutes a serious threat to the poplar plant, a model tree in forest research. The control of this disease by use of chemical fumigants is associated with unwanted side effects such as environmental pollution and drug resistance. However, poplar canker can be effectively controlled with fengycin, an environmentally friendly bio-surfactant of microbial origin. Fengycin homologues are a series of lipopeptides with variations in both the length and branching of the β-hydroxy fatty acid. This study was aimed at purifying and characterizing fengycin homologues produced by Bacillus subtilis from poplar wood bark. A total of 13 fengycin homologues were obtained through 70% ammonium sulfate precipitation, methanol extraction, ion exchange chromatography, gel filtration chromatography, and semi-preparative high-performance liquid chromatography (HPLC). Electro-spray ionization mass spectrometry (ESI-MS) and electro-spray ionization tandem mass spectrometry (ESI-MS/MS) were employed to elucidate the chemical structures of these compounds, and the structures were further characterized by amino acid analysis. Thirteen (13) of the active compounds were fengycin A or B homologues. All the 13 homologues exhibited antifungal activity against the indicator strain Botryosphaeria dothidea. This finding is considered significant, in that it is the first report on the production of so many fengycin homologues and isomers by Bacillus subtilis. Thus, B. subtilis from poplar wood bark is a rich source of environment-friendly, anti-microbial agents for control of poplar canker.

Bacterial wilt and canker of tomato: fundamentals of a complex biological system

Tomato (Solanum lycopersicum) is well-known as a model for study of plant–pathogen interactions, since it is a crop of global relevance and susceptible to multiple bacterial, fungal, viral and nematode pathogens. Among bacterial phytopathogens, the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial wilt and canker of tomato, considered a quarantine disease at international level. The tomato–Cmm interaction has been studied to decipher the pathogenicity mechanisms in Cmm, susceptibility mechanisms in tomato, molecular basis of resistance to Cmm in wild species relative to domesticated tomato, and the level of genetic variability in Cmm. The objective of this review is to discuss recent advances in tomato–Cmm compatible interaction, which can be integrated for application in early diagnosis and biological control of bacterial wilt and canker of tomato. Further study of plant–microorganism interactions is a promising field for improvements in tomato pathogen resistance.

Azole sensitivity in Leptosphaeria pathogens of oilseed rape: the role of lanosterol 14\u03b1-demethylase

Lanosterol 14-α demethylase is a key enzyme intermediating the biosynthesis of ergosterol in fungi, and the target of azole fungicides. Studies have suggested that Leptosphaeria maculans and L. biglobosa, the causal agents of phoma stem canker on oilseed rape, differ in their sensitivity to some azoles, which could be driving pathogen frequency change in crops. Here we used CYP51 protein modelling and heterologous expression to determine whether there are interspecific differences at the target-site level. Moreover, we provide an example of intrinsic sensitivity differences exhibited by both Leptosphaeria spp. in vitro and in planta. Comparison of homologous protein models identified highly conserved residues, particularly at the azole binding site, and heterologous expression of LmCYP51B and LbCYP51B, with fungicide sensitivity testing of the transformants, suggests that both proteins are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. Fungicide sensitivity testing on isolates shows that they sometimes have a minor difference in sensitivity in vitro and in planta. These results suggest that azole fungicides remain a useful component of integrated phoma stem canker control in the UK due to their effectiveness on both Leptosphaeria spp. Other factors, such as varietal resistance or climate, may be driving observed frequency changes between species.

Effectiveness of field isolation distance, tillage practice, cultivar type and crop rotations in controlling phoma stem canker on oilseed rape

Modern agriculture has led to simpler agricultural landscapes that favour the spread of pathogens and increase pressure from pests and diseases. Landscape-dependent interactions between crops and pathogens, including disease related dispersal patterns, and the benefits of reducing pathogen significance call for the design of disease-suppressive landscapes. Model-based assessment is the most efficient method of choosing among management strategies. Based on a case study in France, we ranked the effectiveness of different crop mosaics for control of phoma stem canker on winter oilseed rape (WOSR). Assessed crop mosaics were developed from strategies defined by local stakeholders: (1) isolating target from source fields (all WOSR or only WOSR harbouring RlmX specific resistance), and (2) specifying tillage on WOSR stubble according to cultivar type (with or without RlmX). Model simulations highlighted the effectiveness of WOSR-isolation as compared to RlmX-isolation. Our analyses suggest that tillage (mouldboard ploughing) was the most important factor in explaining the size and genetic structure of the pathogen population (determinant in explaining the breakdown of resistance), and yield loss. While the pathogen population and yield loss decreased with intensive management of non-RlmX-cultivars (85% of WOSR), the same management with RlmX-cultivars modified the genetic structure of the pathogen population. Increasing isolation distances led to reductions in pathogen population and yield loss only in the strategy of WOSR-isolation. Isolating source and target RlmX-cultivar had no effect on the evolution of the population's genetic structure. Although effective in phoma stem canker control, changing tillage can require significant changes for farms. Isolation distance would require extensive information on the landscape, and imply an aggregation of crops that might or might not be possible depending on a farm's spatial organization. This study could lead to the design of a Decision Support System targeting high risk (diseased) WOSR fields to be ploughed or isolated from the following year's cultivation.

Production of three new grapefruit cybrids with potential for improved citrus canker resistance

Cybrid production, combining the nucleus of one species with alien cytoplasmic organelles of another, is a potentially valuable method used for improvement of various crops including Citrus species. Furthermore, this technology is considered a non-GMO biotechnology strategy. In citrus, cybrid plants can be produced as a by-product of somatic fusion. Host resistance is the most desirable strategy for control of citrus canker. By using a cybridization approach, several putative cybrids were created by protoplast fusion of embryogenic suspension culture-derived protoplasts of canker resistant ‘Meiwa’ kumquat ( Thunb ), with mesophyll-derived protoplasts of three grapefruit (Citrus paradisi Macfad.) cultivars ‘Marsh,’ ‘Flame,’ and ‘N11-11’ somaclone of ‘Ruby Red.’ In an effort to generate new grapefruit cultivars with enhanced canker resistance, putative cybrid grapefruit plants morphologically equivalent to standard grapefruit from all three combinations were produced. Four mitochondrial (mt) introns, a mt ribosomal RNA spacer region, and four chloroplast (cp) DNA regions previously shown to have polymorphism among different Citrus species were tested. Four molecular markers, two mt DNA regions (intron nad7i2 and a rRNA spacer), and two cp DNA regions (NADH dehydrogenase subunit K (ndhk) gene and a trnG-trnR intergenic spacer) revealed polymorphism between kumquat and grapefruit and were used to validate the cybrids. All the cybrids had the mt genome of kumquat, and most had the cp genome of kumquat with a few exceptions. EST-SSR marker analysis confirmed that the nuclear genome in all the generated cybrids came from the grapefruit parent. All the cybrid clones have been propagated and are undergoing extensive canker assays to identify any clones that have improved canker tolerance/resistance. These cybrid populations provide a valuable tool for investigating the contribution of cytoplasmic organelles to plant disease resistance.

Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease.

Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

Soluble and insoluble copper formulations and metallic copper rate for control of citrus canker on sweet orange trees

Copper bactericides have been widely studied and used for control of citrus canker caused by Xanthomonas citri subsp. citri. New formulations become available periodically, however, little is known about the relative efficacy of insoluble (ICuF) and soluble (SCuF) copper formulations and the performance of ICuF at the same metallic copper rates. In the present study, we compared three rates of five ICuF (syn. fixed copper) and two SCuF for control of citrus canker during two seasons in a commercial orchard of ‘Valencia’ sweet orange located in Paranavaí, Paraná, Brazil. Control of citrus canker was evaluated as the temporal progress of canker incidence on leaves, cumulative dropped fruit with canker, and incidence of diseased fruit at harvest. All copper formulations and rates reduced the progress of citrus canker incidence on leaves. However, SCuF were not as effective as the ICuF. The incidence of leaves with canker on trees treated with ICuF never exceed 7.3%. In contrast, incidence of leaf canker on trees treated with SCuF ranged from 9.9 to 44.8%. Fruit drop per tree due to citrus canker for trees treated with ICuF did not exceed 6.2 and 17.3 in seasons 1 and 2, respectively. These losses were significantly lower than 20.4 and 38.6, observed for the untreated trees. Fruit drop was 10.4 and 20.9 for trees treated with SCuF in seasons 1 and 2, respectively. The citrus canker incidence on harvested fruit followed the same trend observed for other variables assessed. A non-linear regression analysis showed that the metallic copper rate accounted for a large proportion of the variation in incidence of citrus canker on leaves, fruit and premature fruit drop up to 1 kg of metallic copper/ha/application. Rates exceeding l kg/ha of metallic copper did not consistently improve disease control or reduce the fruit loss.