Detection of Citrus Psorosis Virus by ELISA, Molecular Hybridization, RT-PCR and Immunosorbent Electron Microscopy and its Association with Citrus Psorosis Disease

Psorosis is a citrus disease of undemonstrated etiology that can be diagnosed by biological indexing on sweet orange seedlings followed by a cross protection test. Its presumed causal agent is Citrus psorosis virus(CPsV), type species of the genus Ophiovirus. We compared detection of CPsV by ELISA, RT-PCR, molecular hybridization and immunosorbent electron microscopy, and examined its association with psorosis disease in 11 biologically characterized isolates and in 47 uncharacterized field sources by observation of field symptoms and by biological indexing including the cross protection test. Detection of CPsV by any of the four procedures always coincided with diagnosis of psorosis by cross protection, but it did not always correlate with observation of symptoms thought to be specific, in field trees or in graft-inoculated indicator plants. Trials to detect CPsV by ELISA, molecular hybridization and RT-PCR in citrus sources from different geographical origins, presumed to be psorosis-infected on the basis of field symptoms or reaction of indicator plants, were sometimes unsuccessful, indicating that psorosis symptoms may be induced by causes other than CPsV.     

Effects of fruit maturity and wetness on the infection of apple fruit by Neonectria galligena

Neonectria galligena can cause European canker of apple as well as fruit rot. Healthy unwounded fruits on potted trees of cvs Cox, Bramley and Gala were inoculated with conidia of N. galligena to investigate the effects of wetness duration and fruit maturity on rot development. Overall, the incidence of fruit rot was influenced more by fruit maturity at the time of inoculation than by duration of wetness (6–48 h). Young fruit were most susceptible to infection, with 50% of fruit infected when inoculated up to 4 weeks after full bloom. The susceptibility decreased initially until c. 2 months after full bloom and then increased gradually until harvest. Almost all preharvest symptoms (eye rot) developed only on the fruit inoculated up to 4 weeks after full bloom. All other rots were observed after six‐month postharvest storage under controlled atmospheric conditions. However, the relative proportion of preharvest eye rots and postharvest storage rots varied greatly among three years. The effect of wetness duration was only significant for fruit inoculated in their early stages of development but not for those inoculated near harvest. Regression models were developed to describe the observed effects of fruit maturity and wetness on the incidence of total nectria rots.     

Improving sooty blotch and flyspeck severity estimation on apple fruit with the aid of standard area diagrams

Sooty blotch and flyspeck is caused by numerous species of fungi that colonize the surface of apple fruit and thereby lower its market value. Although this disease poses a substantial threat to apple growers’ profitability in some regions, reliable and cost-effective methods for epidemiological and disease control studies have not been validated, nor are they widely available. We modified a standard area diagram to aid sooty blotch and flyspeck severity assessments and quantified its impact on accuracy and precision of visual estimates. Samples of ‘Fuji’ and ‘Mutsu’ fruit were photographed both from the top and laterally. Severity was assessed from a sub-sample of 160 images using image analysis software. Validation of the diagram was performed by eight raters who independently assessed severity in two series of selected images representing the lateral view and the top view, initially unaided and subsequently with the aid of the scale. Severity estimates ranged from 0.4% to 98% (most fruit had <10% severity). Accuracy and precision of the estimates were significantly improved when using the diagrammatic scale; concordance correlation coefficient values increased from 0.81 to 0.95. A strong tendency to underestimate severity for the mid-range to high levels was minimized when using the aid, which also improved reproducibility of the estimates among raters. In addition to strengthening evidence that a standard area diagram can be used reliably in sooty blotch and flyspeck studies, we expanded its application to disease assessment in the peduncle region, which enhances the usefulness of the method for evaluating efficacy of management practices.     

Effect of infection by the shot-hole fungus,Wilsonomyces carpophilus,on drop and quality of almond fruit 1

Almond fruits were inoculated with suspensions containing 10³, 10⁴ or 10⁵ conidia per mL of Wilsonomyces carpophilus beginning when fruits emerged from the hypanthium and continuing at approximately weekly intervals until they reached full size and embryo development began. More small than mid-sized fruits fell and abscission ceased when fruits reached maximum size. Abscission of mid-sized fruits generally increased with increasing inoculum concentration, and lesions were most abundant on mid-sized fruits. The kernel quality of infected and healthy fruits were compared in 1994 and 1995. Mid- to full-sized fruits were inoculated one or three times with 10 conidia per mL of W. carpophilus. Kernels from inoculated and non-inoculated fruits did not differ in weight, length, or percentage of kernels covered with gum deposits or incompletely filled.     

Modelling the effect of cuticular crack surface area and inoculum density on the probability of nectarine fruit infection by Monilinia laxa

The effects of cuticular crack surface area and inoculum density on the infection of nectarine fruits by conidia of Monilinia laxa were studied using artificial inoculations with conidial suspensions and dry airborne conidia during the 2004 and 2005 seasons, respectively. Additionally, the effect of ambient humidity on fruit infection was evaluated in the 2005 experiment. An exploratory analysis indicated that (i) ambient humidity did not significantly explain the observed variability of data, but that (ii) the incidence of fruit infection increased both with increasing inoculum density and increasing surface area of cuticular cracks. The product of these two variables represented the inoculum dose in the cracks, and was used as a predictor of fruit infection in the model. Natural infection in the orchard was observed to increase throughout the season in both 2004 and 2005. The relationship between the probability of fruit infection by M. laxa and the artificially inoculated dose in the cuticular cracks was well described by a logistic regression model once natural inoculum density was taken into account (pseudo R² = 65%). This function could be helpful for estimating the risk of fruit infection at harvest based on fruit size and natural inoculum density.     

Effects of temperature,zoospore concentration,infection period,and fruit maturity on <Emphasis Type="Italic">Phytophthora palmivora</Emphasis> infection of figs

White powdery rot in figs caused by Phytophthora palmivora is an important disease resulting in severe fruit rot, but is not currently effectively controlled in Japan due to a lack of understanding of its epidemiology. Therefore, the effects of temperature, zoospore concentration, infection period, and fruit maturity on infection of figs were examined by inoculating the fruit with a suspension of P. palmivora zoospores. The zoospores germinated at temperatures from 5 to 35 °C, with the optimum temperature range being 20–35 °C. Germ tube length in zoospore cysts was greatest at 20–30 °C. The disease developed in green figs at temperatures from 20 to 30 °C. Figs inoculated with as few as 10 zoospores per fruit developed severe symptoms at the optimum temperature (25 °C). The minimum infection period required for infection was 2 h at 20–28 °C. All of the figs developed symptoms within an 8 h infection period at 25 or 28 °C, and with a 6 h infection period at 25 °C. All fruit at different stages of development (immature fruit, yellow fruit, and mature fruit) developed symptoms. These results indicate that P. palmivora is capable of infecting figs over a wide range of temperatures, within a short infection period, at a low concentration of zoospores, and at any stage of development. These data could be used to construct forecasting models and develop effective control systems for white powdery rot.     

Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents

A non-radioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd) diagnosis has been developed. The high sensitivity and specificity of this RNA probe in dot blot hybridizations to nucleic acids from field samples, allowed the confirmation of the presence of HSVd in apricot (Prunus armeniaca L.) and its detection in two fruit tree species not previously described as hosts of this pathogen, almond (Prunus dulcis Miller) and pomegranate (Punica granatum L.). This result supports and extends the notion of the world wide distribution of HSVd, infecting cultivated fruit trees. HSVd was also found to accumulate to much higher levels in mature apricot fruits than in leaves. Additionally, a sample processing procedure which does not involve the use of organic solvents was demonstrated to render faithful results when used for viroid detection. The combined reliability and facility of use of both this extraction procedure and the non-radioactive probe will benefit agronomic investigations addressing the detection and eradication of HSVd. Other applications of the work described here, as the study of possible causal relations between specific disorders and HSVd infection, are also discussed.     

Detection of Meloidogyne enterolobii in potatoes in South Africa and phylogenetic analysis based on intergenic region and the mitochondrial DNA sequences

Root-knot nematodes (Meloidogyne spp.) are a major problem facing crop production globally including potatoes. During the 2011/2012 potato growing season, root-knot nematode infected potato tubers were obtained from different potato growing regions in South Africa for identification of Meloidogyne spp. Using the intergenic region of the ribosomal DNA (IGS-rDNA) together with the region between the cytochrome oxidase small subunit II (COII) and the 16S rRNA gene in the mitochondrial DNA (mtDNA), five of the 78 composite samples received produced amplicon sizes of 705 bp for COII and 780 bp for IGS typical of M. enterolobii. These five samples were from the KwaZulu-Natal potato producing region. Nucleotide sequencing and phylogenetic analysis of the COII and IGS fragment showed that the five Meloidogyne populations were 100 % similar and they clustered closely with those of M. enterolobii in the GenBank database. The high damage potential of resistance-breaking populations of Meloidogyne species is a threat to profitable potato production and will require effective pest management programmes to be put in place.     

Field models for the prediction of leaf infection and latent period of Fusicladium oleagineum on olive based on rain,temperature and relative humidity

Although much is known about the effect of climatic conditions on the development of peacock leaf spot of olive, field‐operational models predicting disease outbreaks are lacking. With the aim of developing such models, a 10‐year survey was conducted to relate leaf infection to climate parameters that can be easily monitored in the field. As outbreaks of disease are known to be linked to rain, models were evaluated for their ability to predict whether infection would occur following a rain event, depending on air temperature and duration of relative humidity above 85%. A total of 134 rain events followed by confirmed leaf infection and 191 rain events not followed by detectable infection were examined. The field data were adequately fitted (both specificity and sensitivity >0·97) with either a multilayer neural network or with two of six tested regression models describing high boundary values of high humidity duration, above which no infection occurred over the temperature range, and low boundary values below which no infection occurred. The data also allowed the selection of a model successfully relating the duration of latent period (time between infection and the first detection of leaf spots) as a function of air temperature after the beginning of rain (R² > 0·98). The predictive abilities of these models were confirmed during 2 years of testing in commercial olive orchards in southern France. They should thus provide useful forecasting tools for the rational application of treatments and foster a reduction in fungicide use against this major disease of olive.     

Influence of Colletotrichum simmondsii R. G. Shives & Y. P. Tan infection on selected primary and secondary metabolites in strawberry (Fragaria x ananassa Duch.) fruit and runners

The effect of Colletotrichum simmondsii infection on the contents of sugars, organic acids, and individual phenolic compounds was investigated in strawberry cultivar ‘Clery’. Primary metabolites were determined with the use of HPLC and secondary metabolites further confirmed with HPLC-MS. Colletotrichum simmondsii caused a decrease in sucrose and an increase in fructose and glucose in strawberry fruit. A significant decrease in the content of malic and citric acids was recorded in infected fruit. 12 forms of ellagic acid, nine flavanols and eight flavonols were identified in strawberry runners and nine forms of ellagic acid, six flavanols, seven flavonols and four anthocyanins in strawberry fruit. Significant differences in individual phenolic compounds in strawberry fruit were detected at the beginning of the infection compared to non-infected fruit. Specifically, ellagic acids significantly increased, flavonols generally decreased, and flavanols and anthocyanins increased with the progression of infection. Similarly, some forms of ellagic acid increased and others decreased in infected runners, procyanidins generally decreased and flavonols, increased but the differences were much less prominent.     

Detection of Pseudomonas syringae pv. actinidiae using polymerase chain reaction (PCR) primers based on the 16S–23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions

Several published polymerase chain reaction (PCR) primers to identify Pseudomonas syringae pv. actinidiae, the causal organism of bacterial canker of kiwifruit, were found not to be specific. Two new sets of PCR primers, PsaF1/R2 and PsaF3/R4, were designed to be complementary to a portion of the 16S–23S rDNA intertranscribed spacer (ITS) regions. These primers amplified a DNA fragment from strains of P. syringae pv. actinidiae, but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen, P. syringae pv. theae. When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections as P. syringae pv. actinidiae, all except six cultures produced the expected product of 280 bp with PsaF1/R2 and 175 bp with PsaF3/R4. Results of multilocus sequence analysis using five housekeeping genes (gyrB, acnB, rpoD, pgi and cts) showed that none of these six strains was phylogenetically similar to P. syringae pv. actinidiae. In contrast to the P. syringae pv. actinidiae type strain, these strains were positive in the determinative tests for ice nucleation and syringomycin production. It is suggested that these six strains were incorrectly identified as P. syringae pv. actinidiae. It was not possible to distinguish P. syringae pv. actinidiae from the phylogenetically similar P. syringae pv. theae using the ITS, gyrB, acnB, rpoD, pgi or cts gene regions to design PCR primers. Because P. syringae pv. theae is unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue.