Overexpression of a Gene Encoding a Catabolite Repression Element in Alternaria citri Causes Severe Symptoms of Black Rot in Citrus Fruit

ABSTRACT A gene (AcCreA) encoding a catabolite repression element (CreA) with (two zinc fingers of the Cys(2)His(2) type was isolated from the postharvest fungal pathogen Alternaria citri. The AcCreA overexpression mutant AcOEC2 of A. citri showed normal growth on pectin medium and on segments of peel or the juice sac area from citrus fruit. Production of endopolygalacturonase, an essential virulence factor of this pathogen, was similar in AcOEC2 and the wild type in pectin-containing media. However, addition of glucose to the medium showed that carbon catabolite repression of endopolygalacturonase gene (Acpg1) expression, as well as endopolygalacturonase production, was lost in AcOEC2. The wild-type strain of A. citri causes rot mainly in the central axis of citrus fruit without development of rotting in the juice sac area; however, AcOEC2 caused severe black rot symptoms in both the central axis and juice sac areas. These results indicate that AcCreA-mediated catabolite repression controls the virulence or infection of this pathogen, and that the wild-type A. citri does not cause symptoms in the juice sac area due to carbon catabolite repression by sugars in the juice of the juice sac area.     

Green Fluorescent Detection of Fungal Colonization and Endopolygalacturonase Gene Expression in the Interaction of Alternaria citri with Citrus

ABSTRACT Alternaria citri, a postharvest pathogen, produces endopolygalacturonase (endoPG) and causes black rot on citrus fruit. We previously described that an endoPG-disrupted mutant of Alternaria citri was significantly reduced in its ability to macerate plant tissue and cause black rot symptoms on citrus. In order to investigate colonization of citrus fruit tissues by Alternaria citri, pTEFEGFP carrying a green fluorescent protein (GFP) gene was introduced into wild-type Alternaria citri and its endoPG-disrupted mutant (M60). Green fluorescence was observed in spores, germ tubes, appressoria, and infection hyphae of transformants G1 (derived from wild type) and GM4 (derived from M60). Hyphae of G1 but not GM4 vertically penetrated the peel, but the hyphae of both G1 and GM4 spread equally in the juice sac area of citrus fruit. Green fluorescence of Alternaria citri transformant EPG7 carrying a GFP gene under control of the endoPG gene promoter of Alternaria citri was induced by pectin in the peel during the infection stage, but repressed completely in the juice sac area, likely by carbon catabolite repression by sugars in the juice.     

Wind speed and wind-associated leaf injury affect severity of citrus canker on Swingle citrumelo

Citrus canker (caused by the bacterial pathogen Xanthomonas citri subsp. citri, Xcc) can cause severe damage to citrus. It is endemic in Florida, and occurs in other citrus growing regions. The bacterium is dispersed predominantly in rain splash. To simulate dispersal in splash, and to investigate the effect of wind speed on infection, young plants of Swingle citrumelo were exposed to sprayed inoculum at different wind speeds. Wind was generated using an axial fan, and a pressurized sprayer delivered the inoculum spray. In the five experiments, higher wind speeds (>10 m s⁻¹) consistently resulted in higher incidence and severity of citrus canker developing. By 15 ms⁻¹, there was a dramatic increase in disease. Visible injury to leaves of Swingle citrumelo due to wind was evident at wind speeds ≥ 13 m s⁻¹. The relationship between wind speed and disease, and wind speed and injury was described by a logistic model. More disease was associated with visible injury as the wind speed increased, and disease not associated with visible injury also increased with wind speed. The petiole-leaflet junction was more often infected at higher wind speeds (≥17 m s⁻¹). The concentration of the Xcc inoculum increased the incidence and severity of citrus canker in all experiments. Reducing wind speed in citrus groves with the aid of wind breaks may contribute to a reduction in the severity of an epidemic by reducing dispersal and infection events.     

Detached leaf inoculation of germplasm for rapid screening of resistance to citrus canker and citrus bacterial spot

A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10⁴, 10⁵, or 10⁸ cfu ml⁻¹ were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10⁸ cfu ml⁻¹), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10⁵ cfu ml⁻¹, populations 14 dpi were <10⁴ per inoculation site. In canker-susceptible Citrus spp. (‘Duncan’ grapefruit and ‘Rough’ lemon), water-soaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10⁵ cfu ml⁻¹ by 14 dpi. In citrus bacterial spot-susceptible citrus (‘Swingle’ citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10⁵ cfu ml⁻¹ were comparable to Xcc in susceptible hosts 14 dpi (>10⁵). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.     

Over-expression of the Arabidopsis <Emphasis Type="BoldItalic">NPR1</Emphasis> gene in citrus increases resistance to citrus canker

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc), is a serious leaf and fruit spotting disease affecting many important citrus cultivars including grapefruit and certain sweet oranges. Currently, efficacious and economical disease control measures for highly susceptible citrus cultivars are lacking. Development of commercial cultivars with greater field resistance to citrus canker is the optimum strategy for effective disease management. In this study, we generated transgenic ‘Duncan’ grapefruit (DG) and ‘Hamlin’ sweet orange (Ham) expressing the Arabidopsis NPR1 gene (AtNPR1), which is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR). Our results indicate that over-expression of AtNPR1 in citrus increases resistance to citrus canker and that the resistance is related with the expression levels of AtNPR1 in the transgenic plants. The line (DG 42-2) with the highest expression level of AtNPR1 was also the most resistant, which developed significant fewer lesions accompanied by a ten-fold reduction in Xcc population. The lesions developed on DG 42-2 were smaller and darker than those on the control and lacked callus formation. These lesion phenotypes resemble those on canker resistant kumquats and canker susceptible citrus trees treated with SAR-inducing compounds. Therefore, over-expression of AtNPR1 in citrus is a promising approach for development of more resistant cultivars to citrus canker.     

Effects of some fungicides on <Emphasis Type="Italic">Isaria farinosa</Emphasis>, and <Emphasis Type="Italic">in vitro</Emphasis> growth and infection rate on <Emphasis Type="Italic">Planococcus citri</Emphasis>

The effects of some fungicides used against citrus diseases, on mycelial growth and conidial germination of Isaria farinosa (Holmsk.) Fries [Sordariomycetes: Hypocreales] and also on the pathogenicity of the fungus on citrus mealybug, Planococcus citri (Risso), were determined. Systemic fungicides such as tebuconazole, penconazole and nuarimol were the most effective as regards both conidial germination and mycelial growth. Protective fungicides such as captan, chlorothalonil, mancozeb and propineb inhibited conidial germination at between 1 and 5 μg ml⁻¹ concentration, but captan, chlorothalonil and propineb did not inhibit the mycelial growth at 5,000 μg ml⁻¹. Mancozeb inhibited mycelial growth between 2,500 and 5,000 μg ml⁻¹. Sulphur and copper oxychloride did not inhibit the fungus even at very high concentrations. Sulphur, copper oxychloride, fosetyl-al, chlorothalonil and carbendazim did not decrease the mortality percentage caused by I. farinosa. Tebuconazole, penconazole and mancozeb were the most effective and respectively reduced the mortality from 83% to 33%, 28% and 30% in the ovisacs, from 81% to 29%, 27% and 29% in the 1st instar larvae, and from 84% to 34% in the adult females.     

Unstable green fluorescent protein for study of Xanthomonas citri subsp. citri survival on citrus

Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus bacterial canker, an important disease for the citrus industry. Studies of Xac survival in environments outside of the lesion performed in the past may have underestimated the viable population because the recovery was based on the ability of the bacterium to grow on culture media. This study monitored survival of Xac that express green fluorescent protein (GFP) in two different forms: the native protein, and a protein that is unstable due to a specific oligopeptide tail targeted by proteases within the bacterium. Transformed strains of Xac were verified to be stable in their expression of GFP and to show no differences in virulence and fitness compared to wild type strains. Evaluation of protein stability confirmed that strains with unstable GFP only expressed and fluoresced in metabolically active cells, and not in dead bacteria. Fluorescence of unstable GFP strains under confocal microscopy was used to track bacterial survival and biofilm formation on leaf and fruit surfaces. After spray inoculation, aggregates of fluorescing cells of unstable GFP strains formed biofilms on leaves and fruit. Bacterial cells that aggregated on the surfaces only survived when protected from desiccation. Aggregation of viable bacteria in biofilms confirms their role in pathogen survival outside of lesions and protection from bactericide treatments in the field or in the fruit disinfection process.     

Description of copper tolerant Xanthomonas citri subsp. citri and genotypic comparison with sensitive and resistant strains

The copper-based products widely used for control of citrus canker may lead to the development of Xanthomonas citri subsp. citri (X. citri) resistant to copper (Cu^R). However, the study of copper sensitivity of X. citri strains from Paraná state, Brazil, did not reveal the existence of Cu^R, but copper tolerant (Cu^T) strains. The aim of this study was to describe for the first time the existence of Cu^T X. citri and compare the genetic determinants that differentiate the Cu^T strains from the sensitive (Cu^S) and Cu^R strains. Cu^T strains supported intermediate concentrations of copper in comparison to Cu^S and Cu^R. Cu^T strains lack the gene clusters copLAB or copABCD responsible for copper resistance in Cu^R strains and the large plasmids (c. ≥200 kb) that normally carry these genes. The nucleotide sequences of chromosomal homologous genes cohLAB, involved in copper homeostasis, were 100% similar in strains of all phenotypes. Cu^T strains differed from Cu^S strains by the higher expression of the homologous chromosomal genes cohA and cohB in the presence of copper. Cu^T X. citri strains are not precursors of Cu^R strains 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 of X. citri and adoption of containment measures only when necessary.     

Genetic structure analysis of strains causing citrus canker in Iran reveals the presence of two different lineages of Xanthomonas citri pv. citri pathotype A*

West Asia has been recognized as a major centre for the diversification of Xanthomonas citri pv. citri, a citrus quarantine pathogen of considerable economic importance. However, little genotyping data is available mainly due to the paucity of microbial resources in this region. Using a comprehensive strain collection, several genotyping techniques and a pathogenicity assay, the status of strains causing Asiatic citrus canker in Iran, an internationally significant citrus‐producing country, was clarified. All strains were genetically related to X. citri pv. citri pathotype A* (i.e. strains with a host range restricted to Mexican lime and related species) but not to pathotype A (i.e. strains with a wide host range among rutaceous species). The findings were based on discriminant analysis of the principal components of MLVA‐31 data and were further confirmed by pathogenicity data. Two genetically, geographically and pathologically separate groups of strains in Iran were identified. One of the groups had never been previously reported anywhere in the world. A very strong genetic structure was found (R_ST = 0·938), consistent with their geographical isolation. Strains from these two groups also differed in terms of their type III effector repertoire. The atypical host range of one of these groups could explain why some Iranian strains had previously been mistakenly identified as pathotype A. This study suggests the absence of invasive pathotype A strains in Iran (known as DAPC 1), which account for most of the economically important outbreaks internationally.     

Lethal and sub-lethal effects of a novel sulfoximine insecticide,sulfoxaflor, against Asian citrus psyllid and its primary parasitoid under laboratory and field conditions

The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most important international pest of citrus because it transmits the bacteria that cause huanglongbing (HLB). HLB limits citrus production globally. We evaluated the toxicity of sulfoxalor against D. citri and its parasitoid, Tamarixia radiata Waterston. Sulfoxaflor was as toxic as imidacloprid to adult D. citri. The LC₅₀ values for sulfoxaflor and imidacloprid were 8.17 and 5.7 µg AI mL^?1, respectively. The LC₅₀ of sulfoxaflor for T. radiata adults was 3.3 times greater than for D. citri adults. Treatment with sulfoxaflor resulted in reduced oviposition, development of nymphs, and emergence of adult D. citri on plants, as compared with controls. The lowest concentration that reduced adult emergence was 0.6 µg AI mL^?1. There was reduced feeding by D. citri adults on leaves treated with sulfoxaflor. The residual toxicity of sulfoxaflor was equivalent to imidacloprid. Under field conditions, formulated sulfoxaflor reduced populations of D. citri compared with untreated controls. Sulfoxaflor is a novel mode of action and is an effective tool for D. citri management.     

d -Alanine-d -alanine ligase gene ( ddl ) of Erwinia chrysanthemi strain EC16 II: analysis of pectate lyase regulation using ddl ? mutant

 Erwinia chrysanthemi (Ech) triggers soft rot disease mainly by secreting pectate lyase (Pel), which is regulated in a complex manner by many regulatory genes. In a previous study, we used a gene dosage method to show that the ddl gene, which encodes d-alanine-D-alanine ligase, reduced Pel production and tissue maceration by Ech strain EC16n. In this study, the ddl ⁻ marker-exchanged mutant was shown to overcome the long growth lag caused by various salts in the growth medium and to increase Pel production over that by EC16n, especially in a medium containing magnesium salts. Thus, ddl seems to regulate Pel production in a negative manner. Because the profiles of a gel shift assay using the pelE promoter region as the target DNA with crude extracts of EC16n and ddl ⁻ mutant were distinguishable, Ddl is thought to affect the binding of other regulatory proteins. Expression of the ddl gene was induced in the medium containing a low-molecular-weight fraction of potato extract, but it was reduced in that containing both polygalacturonic acid (PGA) and the fraction. The repression of ddl expression by PGA should contribute in part to the in planta hyperinduction of Pel. Received: May 15, 2002 / Accepted: June 20, 2002     

N‐acetylcysteine interferes with the biofilm formation,motility and epiphytic behaviour of Xanthomonas citri subsp. citri

Citrus canker is caused by Xanthomonas citri subsp. citri. Bacterial biofilm formation is important in the development of this disease because it is a factor in epiphytic bacterial survival on leaves and in infection. N‐acetylcysteine (NAC), in addition to having antibacterial properties, reduces biofilm formation by a variety of bacteria and was therefore tested for impairing biofilm formation by X. citri. Copper is currently the antimicrobial compound most commonly applied in agriculture to control citrus canker. Therefore, this study also evaluated a possible synergistic effect between NAC and copper to improve the strategy for controlling this phytopathogen. NAC was found to decrease biofilm formation, the production of extracellular polysaccharides and bacterial stickiness. Motility was also affected in the presence of NAC. The best combination of NAC and copper for controlling X. citri was application of NAC followed by copper 48 h later. The concentrations of 6 mg mL^?1 of NAC and 3·5 μg mL^?1 of copper were able to kill X. citri. NAC inhibited the epiphytic behaviour of X. citri on leaves, altering cell growth and the bacterial ability to form biofilms. The addition of copper to cells previously treated with NAC enhanced its bactericidal activity. In conclusion, NAC has antibacterial properties against X. citri, interfering with bacterial growth, motility and biofilm formation. Under epiphytic conditions, NAC made the cells more susceptible to copper by affecting X. citri biofilm formation. This study opens new possibilities for the use of NAC in combination with copper, possibly resulting in more sustainable management of citrus canker.     

Modelling the progress of Asiatic citrus canker on Tahiti lime in relation to temperature and leaf wetness

The combined effect of temperature (15°C, 20°C, 25°C, 30°C, 35°C, 40°C and 42°C) and leaf wetness duration (0, 4, 8 12, 16, 20 and 24 h) on infection and development of Asiatic citrus canker (Xanthomonas citri subsp. citri) on Tahiti lime plant was examined in growth chambers. No disease developed at 42°C and zero hours of leaf wetness. Periods of leaf wetness as short as 4 h were sufficient for citrus canker infection. However, a longer leaf duration wetness (24 h) did not result in much increase in the incidence of citrus canker, but led to twice the number of lesions and four times the disease severity. Temperature was the greatest factor influencing disease development. At optimum temperatures (25–35°C), there was 100% disease incidence. Maximum disease development was observed at 30–35°C, with up to a 12-fold increase in lesion density, a 10-fold increase in lesion size and a 60-fold increase in disease severity.     

Mutational analysis of type III effector genes from <Emphasis Type="Italic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis>

Xanthomonas citri subsp. citri, the causal agent of citrus canker, relies extensively on a type III secretion system for infection by delivering type III effectors into host cells. In the genus Xanthomonas, two major regulators, HrpG and HrpX, are involved in the expression of genes encoding the type III secretion system. Twenty-three candidate type III effectors were identified as targets for analysis. The involvement in pathogenicity of 20 candidate effector genes in X. citri strain 306 (Xcc-306) was investigated using site-directed mutagenesis. Pathogenicity assays in grapefruit of 19 genes using site-directed mutagenesis revealed that none of the mutants demonstrated to have reduced ability to cause canker disease. A mutation in the TAL effector pthA4 ^– resulted in loss of hypertrophy although no changes were observed in bacterial growth in leaves. Mutations in hrpG, hrpX, or hrpA genes displayed a complete loss of pathogenicity. Moreover, all mutants maintained the ability to trigger a hypersensitive response (HR) in non-host tomato. In contrast to previous studies, hrpG ^– , hrpX ^– and hrpA ^– mutants also retained the ability to elicit an HR in tomato, indicating the presence of an Hrp independent elicitor in Xcc-306.     

Ecological control of citrus pests primarily using predatory mites and the bio-rational pesticide matrine

Panonychus citri and Diaphorina citri are serious citrus pests (mites) in many countries. The predatory mite Neoseiulus cucumeris can prey on both P. citri and D. citri. It is necessary to develop a strategy using predatory mites and selective pesticides that can simultaneously control both pests effectively and sustainably. The toxicities of matrine and abamectin to P. citri, D. citri and N. cucumeris were evaluated in the laboratory. Matrine was highly lethal to D. citri and relatively less toxic to P. citri and N. cucumeris. Abamectin was relatively less toxic to D. citri. The results of the field trials demonstrated the ecological control strategy that combined the release of predatory mites and applications of four matrine sprays from June 2011 to November 2011, which provided better control over P. citri than pesticide applications alone with six sprays during the same period. It achieved similar control levels for D. citri. In Matrine + N. cucumeris orchard, the total number of spiders was significantly larger than that in the Pesticides Only orchard, but the total number of predatory mites was lower. It is inferred that primarily natural enemies and matrine together play a role in controlling citrus pests.     

Effects of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>) juice containing allicin on <Emphasis Type="Italic">Phytophthora infestans</Emphasis> and downy mildew of cucumber caused by <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>

The volatile antimicrobial substance allicin (diallylthiosulphinate) is produced in garlic when the tissues are damaged and the substrate allicin (S-allyl-l-cysteine sulphoxide) mixes with the enzyme alliin-lyase (E.C.4.4.1.4). Allicin undergoes thiol-disulphide exchange reactions with free thiol groups in proteins and it is thought that this is the basis of its antimicrobial action. At 50 μg ml^-1, allicin in garlic juice inhibited the germination of sporangia and cysts and subsequent germ tube growth by Phytophthora infestans both in vitro and in vivo on the leaf surface. Disease severity in P. infestans-infected tomato seedlings was also reduced by spraying leaves with garlic juice containing allicin over the range tested (55–110 μg ml⁻¹) with an effectiveness ranging from approximately 45–100%. Similarly, in growth room experiments at concentrations from 50–1,000 μg ml⁻¹, allicin in garlic juice reduced the severity of cucumber downy mildew caused by Pseudoperonospora cubensis by approximately 50–100%. These results suggest a potential for developing preparations from garlic for use in specialised aspects of organic farming, e.g. for reducing pathogen inoculum potential and perhaps for use under glass in horticulture.     

Transient expression of pepper Bs2 gene in Citrus limon as an approach to evaluate its utility for management of citrus canker disease

The pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains containing the avrBs2 avirulence gene in susceptible pepper and tomato. The avrBs2 gene is highly conserved in the Xanthomonas genus and when bacteria lack this gene their growth in a susceptible host is diminished, indicating that the avrBs2 gene product could confer an adaptive advantage to the pathogen. The avrBs2 of Xanthomonas citri subsp. citri (Xcc), cause of citrus canker, shares 96% homology with avrBs2 of Xcv. To evaluate if Bs2 could recognize avrBs2 of Xcc in citrus plants and thereby activate plant defence mechanisms to increase resistance to canker, transient expression experiments were conducted using Agrobacterium tumefaciens in lemon plants subsequently challenged with wildtype Xcc. The results showed that transient expression of Bs2 reduced canker formation in lemon and induced plant defence mechanisms, as shown by callose deposition and PR‐1 expression. Moreover, when an avrBs2 mutant of Xcc was used, no decrease in disease symptoms was observed. This work shows that the Bs2 gene from Solanaceae is functional in lemon, a member of the Rutaceae family. Therefore, Bs2 is a potential candidate gene for stable expression in transgenic citrus plants in order to improve resistance to canker disease.     

Isolation of a Protein Bound to Canker-forming Factor from Citrus Plant

Interaction analysis using affinity analysis (Affinity Sensors, Cambridge, UK) indicated the presence of proteins bound to Apl1, a virulence factor of Xanthomonas campestris pv. citri required for the formation of canker symptom on citrus, in the fraction 25–50% ammonium sulfate of citrus crude extracts. Three proteins of 25, 50 and 110-kD were eluted from an Apl1-affinity column. Western analysis revealed that Apl1 binds specifically to the 25-kD and 110-kD but not to the 50-kD protein. When crude extracts of soybean and of tobacco were applied to the Apl1-affinity column, only faint bands were detected. This result suggests that Apl1 targets exist specifically in citrus plants. The amino acid sequence of the N-terminal of the 25-kD protein was determined, and homology search analysis revealed that this sequence was almost identical to those commonly present in S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT) from several plants. This enzyme is specific to the substrate trans -caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA for lignin formation. Received 4 November 1999/ Accepted in revised form 26 November 1999