Association of the inhibitor of virus replication with resistance mechanisms in pepper cultivars

The kinetics of accumulation of tobacco mosaic virus (TMV) and the S strain of pepper mild mottle virus (PMMV) in resistant and susceptible pepper cultivars was analyzed by enzyme-linked immunosorbent assay (ELISA). Crude protein extracts from noninoculated leaves of the resistant cultivar significantly inhibited TMV replication in tobacco protoplasts. Inoculation with either TMV or PMMV-S caused a slight increase in its inhibitory potency. In contrast, protein extracts from either inoculated or noninoculated leaves of the susceptible cultivar did not inhibit TMV replication in tobacco protoplasts. The protein extracts from the resistant cultivar gave a positive reaction with antibodies against the tobacco inhibitor of virus replication (IVR) in western blotting. The putative role of this IVR-like protein from the resistant cultivar in resistance against TMV is discussed.     

Suppression of powdery mildew in squash by applications of whitewash,clay and antitranspirant materials

The incidence of powdery mildew, caused bySphaerotheca fuliginea (Schlecht. Fr.) Polacci, in squash plants, was suppressed in the field to 50–60% of that in untreated control plants by weekly sprays with whitewash (Loven or Yalbin) or clay. Loven or Yalbin sprays resulted in significant reductions in disease level, more on the adaxial (59–65%) than on the abaxial (38–52%) surface of leaves, and more on young (53–59%) than on old (12–18%) leaves. The addition of 0.1% Dabak (a commercial sticker) to the whitewashes significantly increased their efficiency (22% above that of the controls), the degree of protection being associated with whitewash concentration. Squash leaves were protected from powdery mildew also by weekly sprays of the antitranspirant Vapor Gard, to a level comparable with that achieved by a whitewash plus sticker. However, the protection levels achieved with whitewashes, clays and antitranspirant were lower than with a fungicide (propiconazole). Experiments conducted under controlled conditions resulted in higher rates of escape from infection and a lower rate of hyphal and conidial development on plants and tissues following whitewash, clay and antitranspirant treatments.     

Sex Pheromone Traps for Monitoring the Peach Twig Borer,Anarsia lineatella Zeller: Effect of Pheromone Components,Pheromone Dose,Field Aging of Dispenser,and Type of Trap on Male Captures

The effects of ratio between sex pheromone components, pheromone dose in the dispenser, aging of dispenser in the field, and trap type on trapping efficiency of males of the peach twig borer,Anarsia lineatella Zeller (Lepidoptera: Gelechiidae), were investigated. To attract males, the optimal ratio between pheromonal components in a binary blend containing (E)-5-decenyl acetate (E5-10:Ac): (E)-5-decenol (E5-10:OH) was 72:28 or 83:17. Dosages of 7 or 0.7 mg of the binary blend containing E5-10:Ac: E5-10:OH (72:28) were equally effective in attracting males. The effect of aging of dispenser (Israeli dispensers, loaded with 7 mg pheromone) in the field on trapping efficiency was moderate. Captures in traps baited with 4-week-old septa did not differ from those in traps baited with 1-week-old septa. The fairly slow loss of attractancy exhibited by rubber septa indicates that septa may be used for at least 4–5 weeks. At high population levels, the nonsticky IPS trap was significantly more effective in capturing males than the sticky Pherocon 1C trap; at low populations, however, the Pherocon 1C trap was better.     

Effect of endotoxic compounds isolated fromVerticillium lecanii on the sweetpotato whitefly,Bemisia tabaci

Toxic products extracted by methanol from intact mycelia ofVerticillium lecanii were tested for their toxicity to eggs, larvae, pupae and adults ofBemisia tabaci and additional insects. All stages ofB. tabaci were sensitive to the toxin, with larvae showing the highest, and adults the lowest, sensitivity. Using a 0.5% crude toxin preparation, the mortality rate ofB. tabaci was 88%, 53.5%, 53.2% and 37% for larvae, eggs, pupae and adults, respectively. High sensitivity of adults, which was characterized by rapid paralysis followed by death, was observed inB. tabaci, Myzus persicae, Aphis gossypii, Acyrthosiphon pisum andFrankliniella occidentalis. Other insects,viz., Heliothis peltigera, Maladera matrida, Carpophilus hemipterus andCeratitis capitata, exhibited significantly lower sensitivity. The toxin was partially purified by differential extraction with organic solvents and on silica gel H and Sephadex LH 20 columns. Based on the toxin inactivation by saponification and its mobility in a polar solvent on thin layer chromatography, it is suggested that the toxic products might be phospholipids.     

Sex Pheromone Traps for Monitoring the European Vine Moth,Lobesia botrana: Effect of Dispenser Type,Pheromone Dose,Field Aging of Dispenser,and Type of Trap on Male Captures

The effects of dispenser type, pheromone dose in the dispenser, aging of dispenser in the field, and trap type on trapping efficiency of males of the European vine moth,Lobesia botrana Den. et Schiff. (Lepidoptera: Tortricidae), were investigated. LB Pherocon caps and locally manufactured septa were equally effective in attracting males to traps. Within the range of 0.1 to 100 μg pheromone/dispenser, response of males increased positively with the pheromone dose. Within the first 7 days tested, pheromone loads of 1 or 10 mg/dispenser were significantly less attractive to males than 0.1 mg/dispenser. The effect of aging of the dispenser (loaded with 1 mg pheromone) in the field on trapping efficiency was significant. Captures in traps baited with 4- and 5-week-old septa were significantly lower (by 24%) than those in traps baited with 1-week-old septa. The fairly slow loss of attractancy exhibited by rubber septa indicates that septa may be kept in traps for at least 5 weeks. The release rate of the pheromone from the Israeli dispensers (1 mg loaded) was measured in a flow system. The emission of the pheromone decreased gradually as a function of age, correlating well with the lower trap catches of field-aged septa. Release rates were reduced by 56%, 70% and 84% after 4, 5 and 6 weeks, repectively. The nonsticky IPS trap was as effective in capturing the European vine moth males as the sticky Pherocon 1C trap. The possibility of using the non-sticky, nonsaturating and easy-to-handle IPS traps may lead to better and easier monitoring of the European vine moth populations.     

Seedborne infection ofAscochyta rabiei in chickpea and its transmission to aerial plant parts

Ascochyta blight of chickpea (Cicer arietinum L.), caused byAscochyta rabiei (Pass.) Labrousse, was found to be both externally as well as internally seedborne in nature, with the external infection being predominant. The pathogen was located primarily on or in the seed coat and in very few cases penetrated into the cotyledons and rarely, to the embryo. Seedling infection resulted from both externally as well as internally seedborne inoculum. The mode of fungal transmission from seed to the foliar plant parts was strictly non-systemic in nature.     

Effects of Neem Seed Derivatives on Brown Planthopper Symbiotes

Populations of intracellular symbiotes declined significantly in brachypterous females of the brown planthopper,Nilaparvata lugens (Stål), which were stressed during nymphal development by caging them on IR20 rice plants treated with 3% neem oil, 5% neem seed kernel aqueous extract, or 10% neem cake aqueous extract. In addition, nymphs grew poorly and symbiote populations in prospective females declined, when reared on rice plants grown in soil treated with neem cake; this indicates systemic action of bioac-tive neem constituents. Addition of custard-apple oil to neem oil did not enhance the inhibitory effects. The effects of neem derivatives on the symbiotes may be either host-mediated — possibly through disturbed neuroendocrine homeostasis, or direct — leading to perturbations of host-symbiote equilibrium.     

Enhanced resistance to citrus canker in transgenic sweet orange expressing the sarcotoxin IA gene

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subp. Citri (Xcc), is a serious disease reported in most citrus-producing areas around the world. Although different levels of field resistance to citrus canker have been reported in sweet oranges, they are usually not sufficient to provide adequate control of the disease. Ectopic over-expression of antibacterial genes is one of the potential strategies to increase plant resistance to bacterial diseases. Previous in vitro results showed that sarcotoxin IA, an antimicrobial peptide isolated from the flesh fly (Sarcophaga peregrina), can be efficient to control different plant pathogenic bacteria, including Xcc. Transgenic “Pera” sweet orange (Citrus sinensis [L.] Osbeck) plants constitutively expressing the sarcotoxin IA peptide fused to the PR1a signal peptide from Nicotiana tabacum for secretion in the intercellular space were obtained by Agrobacterium-mediated transformation using thin sections of mature explants. Citrus canker resistance evaluation in leaves of transgenic and non-transgenic plants was performed through inoculations with Xcc by infiltration and spraying. The Xcc population was up to 2 log unit lower in leaves of the transgenic plants compared to those of non-transgenic controls. Incidence of canker lesions was significantly higher in non-transformed controls (>10 lesions/cm²) than in the transgenic plants (<5 lesions/cm²) after injection infiltration or spraying with Xcc inoculum. Accumulation of sarcotoxin IA peptide in sweet orange tissue did not cause any deleterious effects on the growth and development of the transgenic plants, indicating this approach is suitable to provide resistance to citrus canker.     

Soil application of imidacloprid and related SAR-inducing compounds produces effective and persistent control of citrus canker

Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced season-long control of citrus canker caused by Xanthomonas citri sbsp. citri. Imidacloprid is a neo-nicotinoid that breaks down in planta into 6-chloronicotinic acid, a compound closely related to the systemic acquired resistance (SAR) inducer isonicotinic acid. Potted Swingle citrumelo seedlings (Citrus paradisi × Poncirus trifoliata) were treated with imidacloprid and the SAR inducers, isonicotinic acid, and acibenzolar-s-methyl as soil drenches or with acibenzolar-s-methyl as a foliar spray 1week prior to inoculation of immature leaves with X. citri sbsp. citri. Seedlings were re-inoculated four times over a 24-week period. SAR induction was confirmed by expression of the PR-2 gene (β-1,3 glucanase). Soil drenches of imidacloprid, isonicotinic acid, and acibenzolar-s-methyl induced a high and persistent up-regulation of PR-2 gene expression and reduced the number of canker lesions for up to 24 weeks compared to 4 weeks for foliar acibenzolar-s-methyl. Soil applied inducers of SAR reduced canker lesions up to 70% compared with the untreated inoculated plants. Lesions on leaves were small, necrotic, and flat compared to pustular lesions on inoculated untreated plants. Populations of X. citri sbsp. citri per leaf were reduced 1–3 log units in soil-treated plants compared to inoculated untreated plants.     

Bacteria from the citrus phylloplane can disrupt cell–cell signalling in Xanthomonas citri and reduce citrus canker disease severity

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a disease that affects almost all types of citrus crops. Production of particular Xcc pathogenicity factors is controlled by a gene cluster rpf, which encodes elements of a cell–cell communication system called quorum sensing (QS), mediated by molecules of the diffusible signal factor (DSF) family. Interference with cell–cell signalling, also termed quorum quenching, either by signal degradation or over‐production, has been suggested as a strategy to control bacterial disease. In this study, three bacterial strains were isolated from citrus leaves that displayed the ability to disrupt QS signalling in Xcc. Pathogenicity assays in sweet orange (Citrus sinensis) showed that bacteria of the genera Pseudomonas and Bacillus also have a strong ability to reduce the severity of citrus canker disease. These effects were associated with alteration in bacterial attachment and biofilm formation, factors that are known to contribute to Xcc virulence. These quorum‐quenching bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used to treat citrus canker disease.     

Over-expression of the citrus gene CtNH1 confers resistance to bacterial canker disease

Citrus canker is a devastating disease, caused by Xanthomonas axonopodis pv. citri (Xac). It is well established that the NPR1 gene plays a pivotal role in systemic acquired resistance (SAR) in Arabidopsis. In this study, we report the isolation and characterization of an NPR1 homolog from citrus, namely Citrus NPR1 homolog 1 (CtNH1). Sequence alignment and phylogenetic analysis indicate that CtNH1 is closely-related to the Arabidopsis NPR1 gene and its orthologs from rice, grapevine, and cacao. When over-expressed in citrus, CtNH1 confers resistance to Xac and leads to constitutive expression of the pathogenesis-related (PR) gene chitinase 1 (Chi1), suggesting that CtNH1 is orthologous to NPR1.     

Effect of the duration of inoculum exposure on development of citrus canker symptoms on seedlings of Swingle citrumelo

Citrus canker (Xanthomonas citri subsp. citri, Xcc) is one of the most serious diseases citrus in Florida, and elsewhere in the world. The disease causes yield loss and some fresh fruit trade restrictions may apply. Cultural management techniques such as windbreaks may work by not only reducing wind speed, but also reducing the period of exposure of susceptible foliage or fruit to those wind speeds that support infection from incoming inoculum. To investigate the effect of exposure period to inoculum of Xcc, seedlings of canker-susceptible Swingle citrumelo were exposed to sprayed inoculum for increasing periods at different wind speeds. The incidence and severity of citrus canker was assessed. In three experiments the incidence and severity of citrus canker most often increased with longer periods of exposure to inoculum, especially so at wind speeds of ≥16 m/s compared to wind speeds of ≤5 m/s (wind speed also increased disease incidence and severity). Regression analysis demonstrated relationships between period of exposure to inoculum and the percent infected leaves per plant, the number of lesions per plant, the number of lesions per infected leaf, and for the percent of infected leaves with lesions on the petioles at wind speeds of ≥16 m/s (R²?=?0.16–0.72). Due to the effect of inoculum exposure period and wind speed, attempts should be made to minimize exposure of canker-susceptible citrus when wind speed is highest and inoculum is available. Windbreaks should help minimize periods of exposure to splashed inoculum in high winds.     

Molecular characterization of Citrus yellow mosaic badnavirus (CMBV) isolates revealed the presence of two distinct strains infecting citrus in India

Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the etiologic agent of yellow mosaic disease of citrus in India. The disease was initially reported from the southern parts of India and has now spread to other parts of the country. It is a serious disease of sweet orange (Citrus sinensis) in southern India, where it causes significant yield losses. During a recent survey of citrus groves in the Nagpur region, central India, characteristic mosaic symptoms were observed in mandarin orange (Citrus reticulata) and sweet orange. Virus transmission studies, electron microscopy, PCR amplification and sequencing of cloned PCR products from samples showing mosaic symptoms confirmed the presence of a badnavirus. The CMBV–Nagpur isolate could be transmitted to the Rangpur lime (C. limonia) and acid lime (Citrus aurantifolia) by graft inoculation. Sequence analysis of a segment of ORF-III region and intergenic region (IR) of the viral genome revealed that CMBV–Nagpur isolate formed a distinct clade along with some previously reported isolates that are known to infect acid lime and Rangpur lime. CMBV isolates that infect citrus species other than the acid lime and Rangpur lime formed a second clade. Based on the transmission studies and phylogenetic analyses, it was concluded that at least two strains of CMBV exist in India currently.     

A new method for inoculation of fruit with <Emphasis Type="Italic">Guignardia citricarpa</Emphasis>, the causal agent of citrus black spot

Citrus black spot (CBS) is a fungal disease, caused by Guignardia citricarpa, that has a high economic impact on citrus. Although G. citricarpa has been associated with black spot of citrus, an adequate pathogenicity test is still not available. Thus, our objective was to develop and evaluate a simple, safe, and practical pathogenicity test. We used fruits from Pera-Rio and Valencia sweet orange trees from two different orchards, located in the State of São Paulo, Brazil. Inoculation was performed by placing six disks colonized by G. citricarpa, onto the peel of healthy fruits, previously bagged. In the Pera-Rio sweet orange grove, initial symptoms of the false melanose type resulting from the inoculations were observed 55 days after inoculation (dai). In the Valencia grove, initial symptoms also of the false melanose type resulting from the inoculations occurred 73 dai. A total of 92.8% and 86.6% of the Pera Rio and Valencia fruits inoculated, respectively, showed symptoms of CBS. Citrus black spot symptoms were not observed in any of the control fruits.     

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.     

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.     

Molecular characterization and phylogenetic analysis of <Emphasis Type="Italic">Citrus viroid VI</Emphasis> variants from citrus in China

Citrus viroid VI (CVd-VI) is a viroid originally found from citrus and persimmon in Japan. We report here the identification and molecular characterization of CVd-VI from four growth regions of China. A total of 90 cDNA clones from nine citrus cultivars were sequenced. The sequence homologies of the Chinese CVd-VI and the reference sequence (NC_004359) vary from 94.2% to 97%. The sequence homologies among the Chinese isolates were up to 95.2%. Phylogenetic analysis of the 23 CVd-VI variants from China and Japan showed that they were grouped into two clades, one with 20 citrus variants and another with three persimmon variants, regardless of the geographic origins. Therefore, as with Hop stunt viroid, CVd-VI could also be divided into two types, citrus and persimmon types. Sequence alignment showed that most nucleotide changes between the two clades occurred in the P, V and T_L domains, and analysis indicated that these mutations influenced the predicted secondary structures under minimum energy.     

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.     

Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 10² Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.