Control Of Canker Research Articles

Natural Bactericidal Effects of Psoralea glandulosa Essential Oil for the Control of Bacterial Canker and Speck in Tomato

Bacterial canker and bacterial speck are diseases affecting tomato caused by Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato, respectively. These diseases are considered a serious threat with a strong impact on tomato production and marketing worldwide, especially because of their reduced sensitivity to traditional controls. This work reports the chemical composition of the essential oil (EO) of Psoralea glandulosa and investigates its in vitro antimicrobial activity, along with its main compound, against three strains of Clavibacter michiganensis subsp. michiganensis (CmVC533, CmVLC78, and CmVQ59) and one strain of Pseudomonas syringae pv. tomato (Pst). The results indicate that both the EO and bakuchiol have significant antibacterial capacity, especially the EO, which reaches a minimum inhibitory concentration (MIC) between 4–16 µg/mL and 128 µg/mL, and a minimum bactericidal concentration (MBC) between 8–16 µg/mL and 128 µg/mL for the strains of C. michiganensis subsp. michiganensis and P. syringae pv. tomato, respectively. The EO and bakuchiol also had an inhibitory effect when applied directly onto plates seeded with C. michiganensis subsp. michiganensis. Overall, the results from this study should be verified in the near future by in vivo studies.

Pathogenicity, phylogenomic, and comparative genomic study of Pseudomonas syringae sensu lato affecting sweet cherry in California.

Comprehensive identification of phytopathogens and an in-depth understanding of their genomic architecture, particularly virulence determinants and antibiotic-resistant genes, are critical for several practical reasons. These include disease diagnosis, improved knowledge of disease epidemiology, pathogen diversity, and determination of the best possible management strategies. In this study, we provide the first report of the presence and pathogenicity of genomospecies Pseudomonas cerasi and Pseudomonas viridiflava in California sweet cherry. More importantly, we report a relatively high level of resistance to copper among the population of Pseudomonas syringae pv. syringae (47.5%). This implies copper cannot be effectively used to control bacterial blast and bacterial canker of sweet cherries. On the other hand, no isolates were resistant to kasugamycin, an indication that kasugamycin could be effectively used for the control of bacterial blast and bacterial canker. Our findings are important to improve the management of bacterial blast and bacterial canker of sweet cherries in California.

Delving into Chemical Control Options for Bacterial Canker (Clavibacter michiganensis subsp. michiganensis) in Tomatoes: An In-vitro Study

The 2022/2023 tomato crop witnessed the emergence of Clavibacter michiganensis subsp. michiganensis in a tomato field, leading to symptoms resembling bacterial canker. Identification of the suspected bacterium, C. michiganensis subsp. michiganensis, utilized specific primers (CMM5 and CMM6) for PCR reaction, resulting in a 614 bp fragment. Several fungicides and bactericides were tested for their ability to control bacterial growth in Petri dishes. Fungicides and bactericides that completely inhibit the bacterial growth in Petri dishes included benzalkonium chloride (250 mg a.i./L), copper oxychloride (1680 mg a.i./L with 1000 mg metallic copper/L), copper hydroxide (2764 mg a.i./L with 1800 mg metallic copper/L), fluazinam (500 µL a.i./L), difenoconazole + pidiflumetofen (200 + 120 µL a.i./L), cuprous oxide (1344 mg a.i./L with 1200 mg metallic copper /L), mancozeb + famoxadone (1000 + 100 mg a.i./L), mancozeb (4000 mg a.i./L) and metiram + pyraclostrobin (2200 + 200 mg a.i./L). The packaged dose of casugamycin (60 µL a.i./L) failed to completely inhibit C. michiganensis subsp. michiganensis growth, necessitating doses exceeding 140 µL a.i./L for complete inhibition. Only at a dosage of 140 µL a.i./L was there no observable growth on the Petri dish containing YDC. Label doses of casugamycin did not prevent the growth of any bacteria, albeit partially controlling Clavibacter and Pectobacterium populations. At the dose of 140 µL a.i./L, the sole bacterium that proliferated was Xanthomonas hortorum pv. gardneri. The other bacteria were included in this study focusing on Clavibacter solely to understand the effect of certain products on other important bacteria in tomato cultivation. The active ingredients, difenoconazole + pidiflumetofen (200 + 120 µL/L active ingredient) and fluazinam (500 µL/L active ingredient) effectively suppressed C. michiganensis subsp. michiganensis growth. The study indicates that various tested fungicides and bactericides were effective in curbing C. michiganensis subsp. michiganensis growth under laboratory conditions. Nonetheless, efficacy may fluctuate based on dose and specific product used. Further research, including field trials, is imperative to evaluate product efficacy under real-world conditions and devise comprehensive management strategies for tomato bacterial canker control.

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

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.

A simple and effective VIGS system facilitates the control of citrus canker by silencing CsLOB1

Virus-induced gene silencing (VIGS) is a powerful technology for molecular characterization of gene functions in plants and has the potential to prevent and control plant diseases. Unfortunately, VIGS in many woody perennials such as citrus are severely hindered because they have a long juvenile period and are recalcitrant to infection compared to herbaceous plants. Here, we report the development of a simple and effective VIGS system based on citrus leaf blotch virus (CLBV), a virus endemic in most citrus-producing countries but showing no symptoms in most citrus varieties. By Agrobacterium-mediated vacuum-infiltration (AVI), pCLBV201-su245 and pCLBV201-pds391 triggered efficient gene silencing (85%) and silencing phenotypes began to appear at 30 days post infiltration/inoculation (dpi), a similar period for achieving that on herbaceous plants. Moreover, the silencing phenotypes could still be observed four years post inoculation. Further, pCLBV201-lob369 was constructed to silence the gene lateral organ boundaries1 (lob1), encoding a key factor for susceptibility to citrus canker that caused by Xanthomonas citri ssp. citri (Xcc). The resistance evaluation results showed that the lesion area, disease index, and bacterial content in the pCLBV201-lob369 treatment group were decreased by 64%, 14%, and 67%, respectively. This work provides a simple and effective VIGS system for citrus which has the potential to be used for diseases control.

High-quality genome assembly and genetic transformation system of Lasiodiplodia theobromae strain LTTK16-3, a fungal pathogen of Chinese hickory.

Lasiodiplodia theobromae, as one of the causative agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry. Although the biological characteristics of this pathogen and the occurrence pattern of this disease have been well studied, few studies have addressed the related mechanisms due to the poor molecular and genetic study basis of this fungus. In this study, we sequenced and assembled L. theobromae strain LTTK16-3, isolated from a Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Phylogenetic analysis and comparative genomics analysis presented crucial cues in the prediction of LTTK16-3, which shared similar regulatory mechanisms of transcription, DNA replication, and DNA damage response with the other four Chinese hickory trunk canker-associated Botryosphaeria strains including, Botryosphaeria dothidea, Botryosphaeria fabicerciana, Botryosphaeria qingyuanensis, and Botryosphaeria corticis. Moreover, it contained 18 strain-specific protein clusters (not conserved in the other L. theobromae strains, AM2As and CITRA15), with potential roles in specific host-pathogen interactions during the Chinese hickory infection. Additionally, an efficient system for L. theobromae protoplast preparation and polyethylene glycol (PEG) -mediated genetic transformation was firstly established as the foundation for its future mechanisms study. Collectively, the high-quality genome data and the efficient transformation system of L. theobromae here set up the possibility of targeted molecular improvements for Chinese hickory canker control.IMPORTANCEFungi with disparate genomic features are physiologically diverse, possessing species-specific survival strategies and environmental adaptation mechanisms. The high-quality genome data and related molecular genetic studies are the basis for revealing the mechanisms behind the physiological traits that are responsible for their environmental fitness. In this study, we sequenced and assembled the LTTK16-3 strain, the genome of Lasiodiplodia theobromae first obtained from a diseased Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Further phylogenetic analysis and comparative genomics analysis provide crucial cues in the prediction of the proteins with potential roles in specific host-pathogen interactions during the Chinese hickory infection. An efficient PEG-mediated genetic transformation system of L. theobromae was established as the foundation for the future mechanisms exploration. The above genetic information and tools set up valuable clues to study L. theobromae pathogenesis and assist in Chinese hickory canker control.

Bifunctional peptides as alternatives to copper-based formulations to control citrus canker

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial “killer” peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 μg mL−1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 μg mL−1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations.Key points• The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri.• Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane.• Mel-CgDEF protected citrus leaves against citrus canker disease.

Exogenous priming of chitosan induces resistance in Chinese prickly ash against stem canker caused by Fusarium zanthoxyli

Stem canker is a highly destructive disease that threatens prickly ash plantations in China. This study demonstrated the effective control of stem canker in prickly ash using chitosan priming, reducing lesion areas by 46.77 % to 75.13 % across all chitosan treatments. The mechanisms underlying chitosan-induced systemic acquired resistance (SAR) in prickly ash were further investigated. Chitosan increased H2O2 levels and enhanced peroxidase and catalase enzyme activities. A well-constructed regulatory network depicting the genes involved in the SAR and their corresponding expression levels in prickly ash plants primed with chitosan was established based on transcriptomic analysis. Additionally, 224 ZbWRKYs were identified based on the whole genome of prickly ash, and their phylogenetic evolution, conserved motifs, domains and expression patterns of ZbWRKYs were comprehensively illustrated. The expression of 12 key genes related to the SAR was significantly increased by chitosan, as determined using reverse transcription-quantitative polymerase chain reaction. Furthermore, the activities of defensive enzymes and the accumulation of lignin and flavonoids in prickly ash were significantly enhanced by chitosan treatment. Taken together, this study provides valuable insights into the chitosan-mediated activation of the immune system in prickly ash, offering a promising eco-friendly approach for forest stem canker control.

Phyto-extracts Mediated Biological Control of Citrus Canker

Citrus is second most important fruit crop and has significance economic importance in Pakistan. In Pakistan 2.17 million tonnes citrus was produced on area of 1.94 million acre. Citrus is mostly cultivated in Punjab and affected by different types of plant pathogens. Citrus canker is the most harmful disease among the major diseases of citrus which is caused by Xanthomonas axonopodis pv. citri. The copper based fungicides are mostly employed to manage the citrus canker in the field but excessive use of chemicals have negatively impacted the environment and warrant the need to find out organic solutions therefore the current study was aimed at identifying suitable plants extracts for the control of citrus canker bacterium. Isolation of the bacterium X.axonopodis was done through streaking on agar plate technique. Morphological and bio-chemical tests such as gram staining, KoH test, catalase test and starch hydrolysis test were performed to examine the bacteria X.axonopodispv. citri. Plant extracts were evaluated, in vitro, against the X.axonopodis pv. citri through paper disc method which showed the inhibition zone against the disease. The 100 percent extract of garlic, ginger, aloevera, moringa were used used along with the streptomycin as a positive control. The inhibition zone was 11.67mm and 10.83mm due to garlic and moringa extracts respectively while it was 16.67mm in case of positive control. The ginger and aloevera extracts proved ineffective against the citrus canker bacterium.The efficacy of garlic and moringa extracts were also checked against the X. axonopodis pv. citri under glasshouse conditions. The inoculum was sprayed on Kinnow plants, with three replicates, for each extract and data was recorded 15, 20, 25, 30 and 35 days post inoculation (dpi).The recorded disease severity percentage, after 35dpi, on garlic and moringa treated plants were 9.67% and 14.27% respectively while the 27% disease severity was recorded on control (plants sprayed with autoclaved distilled water). The garlic extract proved to be most effective followed by moringa extract which could be used as a potential organic solution to control citrus canker.

Whole-genome sequencing and comparative genomics reveal the potential pathogenic mechanism of Neoscytalidium dimidiatum on pitaya

ABSTRACT Neoscytalidium dimidiatum (class Dothideomycetes ) is a fungus responsible for canker disease in pitaya stems and fruits, leading to significant economic losses. However, little is known about the pathogenesis, family evolution, and genetic variants of this species. In this study, we report a high-quality genome sequence of N. dimidiatum based on the Nanopore sequencing technology platform for sequencing and Hi-C assembly technology for genome assembly. The genome contains 12 chromosomes (2 n = 2× = 12; diploid), with a sequencing depth of 186.1×, encoding 12,349 proteins. Molecular phylogenetic analysis showed that N. dimidiatum is evolutionarily close to Botryosphaeria dothidea . Compared to other fungi, the N. dimidiatum genome contains many carbohydrate-active enzymes and secondary metabolites. Additionally, we predict that N. dimidiatum contains 121 candidate effectors that may play important roles in infection and colonization, promoting pathogenicity in pitaya. Nine of these effectors were confirmed to contain signal peptides and inhibit BAX/INF1-induced necrosis in Nicotiana benthamiana , demonstrating their importance during infection. Finally, we also confirm that N. dimidiatum does not form an appressorium or infection thread but instead infects pitaya via open stomata. In conclusion, the results provide a foundation for future research on N. dimidiatum and the control of pitaya canker. IMPORTANCE Pitaya canker is a significant disease in the pitaya industry in China, causing significant economic losses. Therefore, systematic research on Neoscytalidium dimidiatum , the fungus implicated in pitaya canker, is essential for comprehending the pathogenesis of this disease and developing effective control strategies. We applied comparative genomics to reveal the genetic evolution, metabolic diversity, environmental adaptation, and pathogenicity of N. dimidiatum , providing ideal targets for studies of pathogenesis and molecular targets for fungicide development. Moreover, the systematic study of the N. dimidiatum growth cycle, morphological characteristics, and molecular phylogenetic analysis can promote a comprehensive understanding of its genetic basis.

Innovative Strategy for the Control of Citrus Canker: Inhibitors Targeting the Type III Secretion System of Xanthomonas citri Subsp. citri.

To find potential type III secretion system (T3SS) inhibitors against citrus canker caused by Xanthomonas citri subsp. citri (Xcc), a new series of 5-phenyl-2-furan carboxylic acid derivatives stitched with 2-mercapto-1,3,4-thiadiazole were designed and synthesized. Among the 30 compounds synthesized, 14 compounds significantly inhibited the promoter activity of a harpin gene hpa1. Eight of the 14 compounds did not affect the growth of Xcc, but significantly reduced the hypersensitive response (HR) of tobacco and decreased the pathogenicity of Xcc on citrus plants. Subsequent studies have demonstrated that these inhibitory molecules effectively suppress the T3SS of Xcc and significantly impair the pathogen's ability to subvert citrus immunity, resulting in a reduction in the level of disease progression. As a result, our work has identified a series of potentially attractive agents for the control of citrus canker.

Study on the biocontrol efficiency and underlying antagonistic mechanism of valsa canker by pyrolysis tar from apple wood

Apple valsa canker, caused by V. mali, often leads to tree bark rot and yields decreasing. Wood tar, a by-product of biomass pyrolysis contains rich antimicrobial active substances. It has good bonding and waterproofing properties, which is ideal for the control of apple canker. In this study, wood tar obtained from pyrolysis of waste fruit wood was used as supplied test fungicides to explore the biocontrol efficiency and underlying antagonistic mechanism of wood tar against V. mali. Based on the bacteriostatic test, the field experiment was carried out to study the comprehensive control effects of smearing once or twice different times dilution wood tar a year after disease spots treated in different ways. The results showed that the concentrations of wood tar were positively correlated with the inhibition rate of hypha and residual duration, in which the inhibition rate of both 2 and 5 times dilution reached 100% and the residual duration lasted for 7 days. And the control effect of twice annual application after striking off diseased tree spots in zones Ⅰ and Ⅱ, after crisscrossing scratches on diseased tree spots in zone Ⅱ gradually reduced with the increase of wood tar dilution times. The control effect of applying wood tar stock solution and 2 times dilution solution after three kinds of disease spot treatments were 100%, which were improved by 274.5%, 49.9% and 66.7% respectively compared with the control fungicides. The present study demonstrated that wood tar showed promising prospects for use as a biological control agent against apple valsa canker under field conditions.

RNA-based disease control of citrus canker caused by Xanthomonas: challenges and perspectives

RNA-based disease control of citrus canker caused by Xanthomonas: challenges and perspectives

Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars is the most effective, environmentally friendly and economic approach for disease control. However, citrus traditional breeding is lengthy and laborious. Here, we develop transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating a 97.4% biallelic/homozygous mutation rate. No off-target mutations are detected in the edited lines. Canker resistance of the cslob1-edited lines results from both abolishing canker symptoms and inhibiting Xcc growth. The transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS and are exempted from EPA regulation. This study provides a sustainable and efficient citrus canker control solution and presents an efficient transgene-free genome-editing strategy for citrus and other crops.

Control of Bacterial Canker in Stone Fruit Trees by Chemical and Biological Products

Bacterial canker, caused by Pseudomonas syringae pv. syringae and pv. morsprunorum, is one of the most important diseases of stone fruit trees (Prunus spp.). The pathogen infects buds, flowers, fruitlets, leaves and shoots, from which the disease spreads to the branches, boughs and trunks, causing necrosis and cankers. The efficacy of different chemical and biological products for the control of bacterial canker on stone fruit trees was tested in 2018–2021. The experiments were conducted in sour cherry, plum and sweet cherry orchards in central Poland. Foliar application of the tested preparations was performed three times a season. The biological efficacy of the tested products in the control of bacterial canker was evaluated on sour cherry on the basis of infected leaves and fruits and on plum and sweet cherry on the basis of infected leaves. The highest efficacy was observed for products containing various forms of copper—copper oxide, copper oxychloride and copper hydroxide—as well as fertilizers with copper gluconate and the fungicide Luna Care 71.6 WG (fluopyram and fosetyl-Al). However, the biological preparations were significantly less effective. The conducted studies showed that preparations based on copper gluconate can be a valuable alternative to typical copper fungicides.

Cork harvest planning and climate: High air humidity favors availability of airborne inoculum of Diplodia corticola

The canker disease caused by Diplodia corticola is one of the most important emerging pathologies of cork oak (Quercus suber) in western Europe. The fungus is dispersed by borer insects, although it is also thought that the spores can be dispersed by wind and rain. The aim of this study was to evaluate the presence of airborne inoculum of D. corticola in managed cork oak stands during cork harvesting season. Semi-passive spore traps were set in eight sampling plots in Catalonia (north-eastern Spain) in summer of 2020. Traps were replaced every week and the number of D. corticolas spores per sampling event was estimated based on a specific nested-qPCR protocol. Spatial-temporal distribution of airborne inoculum accumulation along sampling areas was analyzed using generalized additive models (GAMs). The availability of airborne inoculum resulted rather low with noticeable accumulation peaks in some of the sampled areas. The fitted GAM revealed a positive effect of high air humidity during the sampling period on the availability of spores. This study represents the first attempt to model the spore release of this emerging pathogen, and it provides insights for developing D. corticola canker control strategies based on the precise timing of cork harvesting operations.

In vitro and in vivo activity of chemical fungicides and a biofungicide for the control of wood diseases caused by botryosphaeriales fungi in apple and pear

Dieback and canker disease caused by species of Botryosphaeriales affect the productivity of pome fruit orchards. In this study, we evaluated the efficacy of fungicides and biofungicide for controlling canker and dieback diseases in apple and pear using in vitro and field trials. Isolates of Diplodia seriata, D. mutila, and Dothiorella omnivora from apple, and of D. seriata and Aplosporella aquifolii from pear were used. Cercobin® (thiophanate-methyl), followed by Bellis® (pyraclostrobin + boscalid), and Nativo® (tebuconazole + trifloxystrobin) inhibited mycelial growth, conidial germination and pycnidia formation in vitro in all fungal isolates. T. harzianum produced mycelial growth inhibition and mycoparasitism in dual culture assays. Thiophanate-methyl and T. harzianum fully inhibited pycnidia production, while pyraclostrobin + boscalid and tebuconazole + trifloxystrobin were most effective against conidia germination. In the orchard assays, protective sprays of the fungicides on pruning wounds reduced incidence by up to 20%, while T. harzianum and tebuconazole + trifloxystrobin reduced the lesion severity up 37 and 41%, respectively. This study represents the first evaluation of fungicides and biofungicide effectiveness for the control of canker and dieback diseases of apples and pears in Argentina.

Bioactive copper and Bacillus subtilis for the control and resistance induction against citrus canker in sweet orange [Citrus sinensis (L.) Osbeck] orchard establishment

• The biological product based on Bacillus subtilis lineage QST 731 applied both via drench and foliar sprays reduces the incidence of citrus canker in citrus trees. • The application of Bacillus subtilis lineage QST 731 via drench in combination with foliar sprays of copper hydroxide or bioactive copper increases the efficiency in the control of citrus canker. • Foliar applications of Bacillus subtilis lineage QST 731 reduces the incidence of citrus leafminer injure. • The copper-based product with amino acids, the bioactive copper, increases the expression of the pr2 gene, which encodes the protein β−1,3 glucanase. Resistance is the plant ability to prevent or delay the infection and the development of a pathogen. The objective of this study was to determine the efficiency of treatments with chemical and biological products for the control of citrus canker during the establishment of citrus orchards through resistance induction. The treatments consisted of a combination of foliar and drench applications of products based on Bacillus subtilis (Bs), bioactive copper (BioCu), copper hydroxide (HCu) and acibenzolar-S-methyl (ASM), at the transplanting of the nursery trees to the field and throughout the first year of establishment of the citrus orchard. Evaluations were carried out on mature branches determining the incidence of citrus canker, defoliation, and citrus leafminer (CLM) injures. The vegetative growth of the citrus trees, i.e. tree height and trunk and canopy diameters, were also determined. Under controlled conditions, studies were carried out to determine the activation of the systemic acquired resistance (SAR) by treatments with ASM and Bs applied via drench, and by BioCu and copper sulfate (SCu) applied by spraying on the leaves of the citrus trees. The expression of the PR-2 gene was evaluated by RT-qPCR at 24, 42, and 72 h, and seven days after the application of the treatments. In the field, the ‘IAPAR 73′ sweet orange trees treated via drench with Bs and foliar applications of Bs + HCu, Bs + BioCu and HCu with and without ASM showed reductions in the incidence of citrus canker. HCu treatments reduced defoliation, while foliar treatments with Bs decreased the incidence of CLM injures. The ‘IAPAR 73′ sweet orange trees treated with ASM showed a reduction in the vegetative growth. The BioCu and Bs products were effective for the control of citrus canker in young citrus trees during the orchard establishment. In addition, the BioCu product increased the expression of the PR-2 gene, which encodes the β-1,3 glucanase, a protein associated with the systemic acquired resistance (SAR) in plants.

Systematic stepwise screening of new microbial antagonists for biological control of European canker

Neonectria ditissima is the causal agent of European canker. This pathogen causes cankers on apple branches and the main stem, which may lead to the loss of the whole tree. Chemical control is the essential component in disease management and no suitable biocontrol products are commercially available. This study aimed at selecting potential microbial antagonists against N. ditissima through a systematic stepwise screening program for the development of a new biocontrol product. A total of 520 potential candidates were isolated from apple branches showing canker symptoms. Important characteristics for application of Microbial Biological Control Agents were tested per each candidate: spore production, spore survival during storage, cold tolerance, drought tolerance and UV-B resistance. Isolates able to germinate and grow at human body temperature were excluded. A total of 252 candidates fulfilled the stablished criteria. All 520 candidates belonged to 44 different taxonomic groups, being the most abundant Alternaria spp. (22.2 %), Aureobasidium pullulans (16.1 %), Cladosporium spp. (9.5 %) and Fusarium spp. (9.0 %). Information on possible pathogenicity and toxicity for humans, animals, plants and the environment and on patents in biocontrol use was collected per each identified species. A total of 158 candidates belonging to species that did not show potential risks or patent conflicts were assessed by their antagonistic behaviour against N. ditissima in bioassays in planta. Each candidate was inoculated in ‘Elstar’ apple branches inoculated with N. ditissima 24 h before. Inoculated branches were incubated at 17 °C, 16 h light per day and RH > 90 %. After four weeks, canker symptom expression was visually assessed. The capacity of the candidates to reduce colonisation of N. ditissima in the branches was evaluated by quantifying N. ditissima DNA concentration using qPCR. Four candidates of Clonostachys rosea showed antagonistic properties; after four weeks of treatment, no canker symptoms or small bark cracks were observed in the inoculated branches and N. ditissima DNA was reduced by 90–99 %. Following them, the branches inoculated with one candidate of Akanthomyces muscarius, A. pullulans and Cladosporium europaeum showed small bark cracks and small swollen bark and N. ditissima DNA was reduced by more than 90 %. The systematic stepwise screening approach was a powerful strategy to find new MBCAs against N. ditissima with antagonistic properties that also fulfilled major criteria with regards to commercial production and safety, as well as ecological needs.

Inhibition of the Citrus Bacterial Pathogen Xanthomonas axonopodis pv. citri through Aloysia gratissima Essential Oil and Flourensia spp. Extracts and Prenylflavonoids

Background: Xanthomonas axonopodis pv. citri is a gram-negative bacterium that affects citrus crops, causing a disease known as citrus canker. Although essential oils and other compounds isolated from plants represent a natural alternative to treat this disease, they have the disadvantage of having low solubility in the media in which the bioassays to determine antimicrobial activity are performed. This has led several researchers to evaluate the solubility of plant essential oils in alternative solvents. Objectives: The aim of this study was to evaluate the solubility of the essential oil from Aloysia gratissima as well as that of low-polarity extracts and pure compounds of the genus Flourensia in diluted agar/Tween 80 solutions to test and improve their antimicrobial activity against Xanthomonas axonopodis pv. citri. Methods: Antimicrobial activity against Xanthomonas axonopodis pv. citri was determined by bioautography, agar diffusion, and microdilution methods. Results: The A. gratissima oil showed increased activity in the agar (0.15% m/v)/Tween80 (0.5% v/v) 1:1 mixture, with MIC values ranging from 75 to 100 μL/mL, while Flourensia spp. extracts were more soluble in agar solution (0.15% m/v). The pure compounds tested presented MIC values ranging from 50 to 150 μg/mL. Conclusion: The proven antimicrobial activity of both Aloysia gratissima essential oil and Flourensia spp. extracts and pure compounds allows proposing these natural products as potential antimicrobial agents in the control of citrus canker.