Characterization of electrical penetration graphs of Bucephalogonia xanthophis, a vector of Xylella fastidiosa in citrus

The sharpshooter Bucephalogonia xanthophis (Berg) (Homoptera: Cicadellidae) is a vector of the xylem-limited bacterium, Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner), which causes citrus variegated chlorosis. Despite the importance of citrus variegated chlorosis, the probing behavior of vectors on citrus and its implications for transmission of X. fastidiosa have not been studied. Here we studied electrical penetration graph (EPG-DC system) waveforms produced by B. xanthophis on Citrus sinensis (L.) Osbeck (Rutaceae), and their relationships with stylet activities and xylem ingestion. Electrical penetration graph waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration on plant tissues. The main waveforms were correlated with histological observations of salivary sheaths in plant tissues and excretion analysis, in order to determine stylet activities and their precise position. Six waveforms and associated activities are described: (S) secretion of salivary sheath and intracellular stylet pathway, (R) resting during stylet pathway, (Xc) contact of stylets with xylem vessels, (Xi) active xylem ingestion, (N) interruption within the xylem phase (during Xc or Xi), and (W) withdrawal of stylet from the plant. The sharpshooter spent 91.8% of its probing time with its stylet in the xylem, where the main activity was ingestion (Xi: 97.5%). During a probe, the most likely sequence of events is secretion of salivary sheath and pathway (S) through epidermal and parenchyma cells (all individuals), followed by contact with xylem (Xc) (67.6% of all individuals) and ingestion (Xi) (88.3% of those that exhibit waveform Xc). The mean time to contact the xylem (Xc) and initiate ingestion (Xi) after onset of the first probe was 27.8 and 34.2 min, respectively. However, sustained xylem ingestion (Xi > 5 min) was established after 39.8 min, on average. This information is basic for future studies on the transmission mechanisms of X. fastidiosa and in order to establish control strategies aimed at interfering with this process.

Transmission efficiency of Xylella fastidiosa by sharpshooters (Hemiptera : Cicadellidae) in coffee and citrus

Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) is a bacteria] pathogen transmitted by several Sharpshooters in two tribes of Cicadellinae (Proconiini and Cicadellini). Here, we compared the transmission efficiency of X. fastidiosa in coffee (Coffea arabica L) and citrus [Citrus sinensis (L) Osbeck] by Cicadellini [Bucephalogonia xanthophis (Berg) and Dilobopterus costalimai Young] and Proconiini [Homalodisca ignorata Melichar and Oncometopia facialis (Signoret) I sharpshooters that Occur in both crops. At different seasons, healthy adults of each species were submitted to a 48-h acquisition access period on citrus or coffee source plants infected with X. fastidiosa isolates that cause Citrus variegated chlorosis (CVC) and Coffee leaf scorch (CLS), respectively, and then confined on healthy seedlings of the corresponding host plant for a 48-h inoculation access period. No significant effect of inoculation season was observed when comparing infection rates of citrus or coffee plants inoculated by vectors at different times of the year. In Citrus, the transmission rate by single insects was significantly higher for H. ignorata (30%) in relation to B. xanthophis (5%) and O. facialis (1.1%) but there was no difference among vector species in coffee, whose transmission rates ranged from 1.2 to 7.2%. Comparing host plants, H. ignorata was more effective in transmitting X. fastidiosa to citrus (30%) in relation to coffee (2.2%), whereas the other vectors transmitted the bacterium to both hosts with similar efficiencies. Despite these variations. vector efficiency in coffee and Citrus is lower than that reported in other hosts.

Genetic structure and biology of Xylella fastidiosa strains causing disease in citrus and coffee in Brazil

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.

Effectiveness of two systems for the spraying of pesticides in citrus trees

Citriculture normally uses high application volumes in pesticide solutions (of 2.000 to 5.000 L ha(-1)) to control pests and diseases that affect the crop, which generates an increase in operational costs. For this reason, diverse systems of application are being developed to reduce application volumes and improve the uniformity of pesticide deposition. The goal of this work was to evaluate the efficiency of two application systems of pesticides in citrus trees. One system used a prototype for terrestrial application with rotary disc atomizers that are widely used in agricultural aviation, and the other system used hollow cone tip hydraulics. For the treatment of the trees the insecticide Metidation was used at the dose of 180 gr per hectare. To study the droplet spectrum, water-sensitive papers were installed at different positions in the trees canopy, and for the study of insecticide deposition leaves of the treated plants were collected. The water-sensitive papers were collected and analyzed using a computerized image analysis system (e-Sprinkle, EMBRAPA, Sao Paulo, Brazil), and the leaves analyzed by the technique of gas chromatography. Pesticide deposition was similar in both application system, although the solution volume used by the application system equipped with rotary disc atomizers was one quarter of the volume used by the application system equipped with hydraulic tips, reducing considerably the cost of the phytosanitary treatments.

Irrigation requirements and transpiration coupling to the atmosphere of a citrus orchard in Southern Brazil

Crop evapotranspiration (ETc) was measured as evaporative heat flux from an irrigated acid lime orchard (Citrus latifolia Tanaka) using the aerodynamic method. Crop transpiration (T) was determined by a stem heat balance method. The irrigation requirements were determined by comparing the orchard evapotranspiration (ETc) and T with the reference evapotranspiration (ETo) derived from the Penman-Monteith equation, and the irrigation requirements were expressed as ETc/ETo (Kc) and T/ETo (Kcb) ratios. The influence of inter-row vegetation on the ETc was analyzed because the measurements were taken during the summer and winter, which are periods with different regional soil water content. In this study, the average Mc values obtained were 0.65 and 0.24 for the summer and winter, respectively. The strong coupling of citrus trees to the atmosphere and the sensitivity of citrus plants to large vapor pressure deficits and air/leaf temperatures caused variations in the Kcb in relation to the ETo ranges. During the summer, the Kcb value ranged from 0.34 when the ETo exceeded 5 mm d(-1) to 0.46 when the ETo was less than 3 mm d(-1). (C) 2011 Elsevier B.V. All rights reserved.

Isolation and characterization of microsatellite loci in Colletotrichum acutatum, the causal agent of postbloom fruit drop on citrus

From a genomic enriched library, we developed 27 primer pairs from microsatellite flanking sequences for Colletotrichum acutatum, associated to postbloom fruit drop disease on citrus. Loci were characterized using 40 monosporic C. acutatum isolates. Nine primer pairs successfully amplified polymorphic microsatellite regions, with 3-6 alleles per locus, and mean heterozygosities ranging 0.093-0.590 across loci. The suitability of these primers was investigated in four Colletotrichum species as well. These microsatellite markers will be useful for genetic analyses and epidemiological studies of C. acutatum.

Host status of citrus rootstocks for the population K5 of Pratylenchus jaehni

Host status of citrus rootstocks for the population K 5 of Pratylenchus jaehni Pratylenchus jaehni has been reported as the main nematode in Brazilian citrus orchards due to the economic losses it causes. Considering the reports of differences and similarities in populations from that species, the host reaction of citrus rootstocks to K 5 population of P. jaehni was evaluated and compared with topotypes of the species. Two experiments were carried out with initial population (Pi) of 180 and 200 specimens per plant. In each experiment, the evaluation was carried out in two periods: at 120 and 245 days after inoculation (DAI) in experiment 1 and at 60 and 240 DAI in experiment 2. The variables obtained were the reproduction factor [RF = final population (Pf)/Pi)] and nematodes per gram of roots (N/g). The nematode density was reduced (RF = 0.00 to 0.02) in trifoliata `Limeira`, sour orange, citrange `Carrizo`, `Cleopatra` tangerine, `Sunki` tangerine, `Volkameriano` lemon and citrumelo `Swingle`; but the nematode density increased (RF = 1.89 to 22.28) in Rangpur lime in both periods of experiment 1 and in the second period of experiment 2. Therefore, the host reaction of citrus rootstocks to K 5 was very similar to that reported to topotypes of P. jaehni in the literature.

Annual and polyetic progression of citrus canker on trees protected with copper sprays

The effects of copper sprays on annual and polyetic progress of citrus canker, caused by Xanthomonas citri subsp. citri, in the presence of the Asian citrus leafminer (Phyllocnistis citrella), were evaluated in a study conducted in a commercial orchard in northwest Parana state, Brazil, where citrus canker is endemic. Nonlinear monomolecular, logistic and Gompertz models were fitted to monthly disease incidence data (proportion of leaves with symptoms) for each treatment for three seasons. The logistic model provided the best estimate of disease progress for all years and treatments evaluated and logistic parameter estimates were used to describe polyetic disease dynamics. Although citrus canker incidence increased during each of the seasons studied, it decreased over the whole study period, more so in copper-treated trees than in water-sprayed controls. Copper treatment reduced disease incidence compared with controls in every year, especially 2004-2005, when incidence was ca. 10-fold higher in controls than in treated plots (estimated asymptote values 0 center dot 82 and 0 center dot 07, respectively). Copper treatment also reduced estimated initial disease incidence and epidemic growth rates every year.

Liberibacters associated with orange jasmine in Brazil: incidence in urban areas and relatedness to citrus liberibacters

Two surveys (2005/2006 and 2009) were conducted in the state of Sao Paulo, Brazil, to investigate the incidence of `Candidatus Liberibacter asiaticus` and `Ca. L. americanus`, two liberibacters associated with citrus huanglongbing (HLB) disease and both transmitted by Diaphorina citri, in orange jasmine (Murraya exotica), a widespread ornamental tree in cities and villages. The graft-transmissibility of the two species, and their DNA relatedness to citrus-associated liberibacters, were also investigated. Quantitative PCR was applied to PCR-positive orange jasmine and HLB-positive citrus growing in backyards and orchards to assess their inoculum source potentials. Liberibacters were detected in 91 of 786 sampled orange jasmine plants in 10 of 76 sampled locations. PCR-positive trees exhibited yellow shoots and/or dieback symptoms indistinguishable from those on PCR-negative trees. `Candidatus Liberibacter americanus` was more common in 2005/2006 (96 center dot 6%) and `Ca. L. asiaticus` in 2009 (84 center dot 8%). rplJ nucleotide sequences were identical within all populations of either species. Graft transmission succeeded only in homologous host combinations, including `Ca. L. americanus` (2/10) from/to orange jasmine and `Ca. L. americanus` (5/18) and `Ca. L. asiaticus` (5/9) from/to citrus. Symptoms were mild and developed less rapidly in orange jasmine than in citrus, probably as a result of lower liberibacter multiplication rates. Respective titres of `Ca. L. americanus` and `Ca. L. asiaticus` in orange jasmine averaged 4 center dot 3 and 3 center dot 0 log cells g-1 tissue, compared with 5 center dot 5 and 7 center dot 3 in citrus. The results indicate that orange jasmine does not favour liberibacter multiplication as much as citrus. However, its importance in HLB epidemics should not be underestimated as it is a preferred host of D. citri and is not under any strict tree-eradication programme or measures for insect control.

Probable consequences of the mitigation of citrus canker eradication methodology in Sao Paulo state

Probable consequences of the mitigation of citrus canker eradication methodology in Sao Paulo state Recently the Sao Paulo state government mitigated its citrus canker eradication methodology adopted since 1999. In April 2009 at least 99.8% of commercial sweet orange orchards were free of citrus canker in Sao Paulo state. Consequently the mitigation of the eradication methodology reduced the high level of safety and the competitiveness of the citrus production sector in Sao Paulo state, Brazil. Therefore we suggest the re-adoption of the same eradication methodology of citrus canker adopted in Sao Paulo from 1999 to 2009, or the adoption of a new methodology, effective for citrus canker suppression, because in new sample surveys citrus canker was detected in >0.36% of affected orchards. This incidence threshold was calculated by using the Duncan test (P <= 0.05) to compare the yearly sample surveys conducted in Sao Paulo state to estimate citrus canker incidence between 1999 and 2009. The calculated minimum significant level was 0.28% among sample surveys and the lowest citrus canker incidence in Sao Paulo state was 0.08%, occurring in 2001. Thus, as an alternative, we suggest the adoption of a new eradication methodology for citrus canker suppression when a new sample survey detected >0.36% of affected orchards in Sao Paulo state, Brazil.

Postharvest harpin or Bacillus thuringiensis treatments suppress citrus black spot in `Valencia` oranges

Citrus black spot (CBS) caused by Guignardia citricarpa represents an important threat to citriculture in Brazil. Limited information is available regarding potential biological control agents and new alternative compounds that may provide protection of orange fruits against the disease. In this study, the effects of commercial products based on Bacillus thuringiensis var. kurstaki (Bt) bacterium, Bt pure isolates and Harpin protein (Messenger (R)) on the postharvest control of CBS, were evaluated in `Valencia` sweet orange fruits harvested for three consecutive years in a citrus grove. The fruits were sprayed with the following products: DiPel (R) WP (Bt, subspecies, kurstaki strain HD-1,16,000 International Units mg(-1), 32 g active ingredient kg(-1)) (1, 20 and 50 mg ml(-1)), Dimy Pel (R) WP (Bt, subspecies, kurstaki, strain HD-1, 17,600 IU mg(-1), 26 g active ingredient l(-1)) (2, 20 and 50 mg ml(-1)), Messenger (R) (3% harpin protein) (1 and 2 mg ml(-1)) and fungicide Tecto (R) Flowable SC (thiabendazole, 485 gl(-1)) (0.8g active ingredient l(-1)), besides the Bt isolates, Bt- HD-567, Bt- DiPel and Bt- Dimy (9 x 10(8) CFU ml(-1)). Ten days after treatment, the number of newly developed CBS lesions and pycnidia produced were evaluated using fifty fruits per treatment. The Dimy Pel (R) and Messenger (R) reduced the number of new developed CBS lesions on fruits in up to 67% and 62%, respectively. All applied treatments drastically decreased the number of pycnidia produced in the CBS lesions on orange fruits with 85% to 96% reductions compared to the untreated control. Volatile compounds produced by the isolates Bt- HD-567, Bt- Dimy and Bt- DiPel, reduced the number of lesions on treated fruits by 70%, 65% and 71% compared to the control, respectively. In addition, the survival of Bt isolates on orange fruit surfaces were evaluated by recovering and quantifying the number of CFU every seven days for up to 28 days. The declines in survival rates on orange fruit surfaces were drastic for the three strains of Bt in the first week. The CFU numbers of all applied isolates declined by 4 to 5 orders of magnitude after storage at room temperature for 28 days. In vitro assays revealed that the Bt isolates significantly reduced the mycelial growth of the pathogen, ranging from 32% to 51%, compared to the control, whereas no inhibitory effect was observed in the presence of Messenger (R). (C) 2010 Elsevier Ltd. All rights reserved.

Volatile organic compounds produced by Saccharomyces cerevisiae inhibit the in vitro development of Guignardia citricarpa, the causal agent of citrus black spot

Due to the low chemical control effectiveness of citrus black spot, caused by the fungus Guignardia citricarpa at postharvest, and to the search for alternative control methods, this study aimed to evaluate the in vitro effect of volatile organic compounds (VOCs), produced by yeast Saccharomyces cerevisiae, on G. citricarpa. It was observed that the yeast strains evaluated acted as antagonists by VOC production, whose maximum inhibitory capacity was as high as 87.2%. The presence of fermentable carbon sources in the medium was essential for the bioactive VOC production by the yeast. The analysis of VOCs produced in PDA medium by SPME-GC-MS indicated the presence of high quantities of alcohols as well as esters. An artificial VOC mixture prepared on the basis of the composition of the VOCs mimicked the inhibitory effects of the natural VOCs released by S. cerevisiae. Thus, the VOCs produced by the yeast or the artificial mixtures can be a promising control method for citrus black spot or others postharvest diseases.

Colletotrichum gloeosporioides, a new causal agent of citrus post-bloom fruit drop

Citrus post-bloom fruit drop (caused by Colletotrichum acutatum) frequently occurs in the southwestern region of So Paulo State, Brazil. A survey of Colletotrichum isolates associated with symptoms of post-bloom fruit drop in So Paulo State showed C. gloeosporioides in addition to C. acutatum. The objectives of this study were to confirm the identification of C. gloeosporioides isolated from symptomatic citrus flowers, to test the pathogenicity of C. gloeosporioides isolates, to compare the development of disease caused by C. gloeosporioides and C. acutatum, and to determine the frequency of C. gloeosporioides in a sample of isolates obtained from symptomatic flowers in different regions of So Paulo State. Through the use of species-specific primers by PCR, 17.3% of 139 isolates were C. gloeosporioides, and the remaining 82.7% were C. acutatum. The pathogenicity tests, carried out in 3-year old potted plants of sweet oranges indicated that both species caused typical symptoms of the disease including blossom blight and persistent calyces. Incubation periods (3.5 and 3.9 days, respectively, for C. acutatum and C. gloeosporioides) and fruit sets (6.7 and 8.5%, respectively for C. acutatum and C. gloeosporioides) were similar for both species. The incidences of blossom blight and persistent calyces were higher on plants inoculated with C. acutatum than in those inoculated with C. gloeosporioides. Conidial germination was similar for both species under different temperatures and wetness periods. Under optimal conditions, appressorium formation and melanisation were higher for C. gloeosporioides than for C. acutatum. These results indicated that Colletotrichum gloeosporioides is a new causal agent of post-bloom fruit drop.

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

The combined effect of temperature (15A degrees C, 20A degrees C, 25A degrees C, 30A degrees C, 35A degrees C, 40A degrees C and 42A degrees 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 42A degrees 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 (25-35A degrees C), there was 100% disease incidence. Maximum disease development was observed at 30-35A degrees 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.

Incidence of black spot in citrus fruits from different processing stages in packinghouses and from Sao Paulo`s wholesale market

The purposes of this work were a) to evaluate citrus black spot (CBS) incidence in `Valencia` oranges and `Murcott` tangors aimed at the export market, and in Pera`, `Lima` and `Natal` oranges, and `Murcott` tangors, aimed at the domestic market after different processing stages in packinghouses in 2004/05 and 2005/06; b) to evaluate CBS incidence in Pera` and `Lima` oranges and `Murcott` tangors sold at Ceagesp-SP, the biggest wholesale market in the State of Sao Paulo, in 2006. Citrus fruits were collected at the packinghouse, on their arrival, after pre-washing and de-greening, from the packing table, from the pallet and at Ceagesp. They were stored for 14 to 21 days at 25 degrees C and 85-90% RH. The incidence of CBS was visually evaluated after one day and at the end of the storage period. CBS incidence in fruits aimed at the export market decreased, with values under 2.0% on arrival and no CBS symptoms observed on fruits from the pallet. The average incidence of CBS in `Pera`, `Lima` and `Natal` oranges, and `Murcott` tangors in the packinghouse aimed at the domestic market were 64.1, 39.0, 32.1 and 19.3%, respectively, after one day of storage, then remaining constant in all processing stages. The incidence of CBS in Ceagesp fruits was low in winter months and increased in the spring. The increase in disease incidence during the storage period (21 days) was not significant in collected fruits.