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.     

Phytoplasmas infecting sour cherry and lilac represent two distinct lineages having close evolutionary affinities with clover phyllody phytoplasma

Phytoplasmas infecting sour cherry and lilac in Lithuania were found to represent two lineages related to clover phyllody phytoplasma (CPh), a subgroup 16SrI-(R/S)C (formerly 16SrI-C) strain exhibiting rRNA interoperon sequence heterogeneity. 16S rDNAs amplified from the cherry bunchy leaf (ChBL) and lilac little leaf (LcLL) phytoplasmas were identical or nearly identical to those of operon rrnA and operon rrnB, respectively, of CPh. There was no evidence of 16S rRNA interoperon sequence heterogeneity in either LcLL or ChBL phytoplasma. Based on collective RFLP patterns of 16S rDNA, ChBL was classified in subgroup 16SrI-R, and LcLL was classified in new subgroup 16SrI-S. The ribosomal protein (rp) gene sequences from LcLL phytoplasma were identical to those of CPh, and strain LcLL was classified in rp subgroup rpI-C. By contrast, rp gene sequences from ChBL phytoplasma differed from those of subgroup rpI-C; based on RFLP patterns of rp gene sequences, ChBL was classified in new rp subgroup rpI-O. Single nucleotide polymorphisms (SNPs), designated here by a new SNP convention, marked members of rp subgroup rpI-C, and distinguished LcLL and CPh from ChBL and other non-rpI-C phytoplasmas in group 16SrI. The results raise questions concerning phytoplasma biodiversity assessment based on rRNA genes alone and encourage the supplemental use of a single copy gene in phytoplasma identification and classification, while drawing attention to a possible role of horizontal gene transfer in the evolutionary history of these lineages.     

Haplotypes of “<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum” suggest long-standing separation

Three haplotypes of the recently discovered bacterium species “Candidatus Liberibacter solanacearum” are described and related to geographic ranges. The first two are associated with Zebra Chip/Psyllid Yellows of potatoes and other solanaceous plants, vectored by the tomato/potato psyllid Bactericera cockerelli in North and Central America and New Zealand. The third is associated with diseased carrots in Finland and vectored by the carrot psyllid Trioza apicalis. The haplotypes are described by SNPs on the 16s rRNA, 16s/23s ISR and 50s rplJ and rplL ribosomal protein genes. These SNPs are inherited as a package across the three genes. Haplotype “a” has been found primarily from Honduras and Guatemala through western Mexico to Arizona and California, and in New Zealand. Haplotype “b” is currently known from eastern Mexico and northwards through Texas to south central Washington. These haplotypes show some range overlap in Texas, Kansas and Nebraska. The haplotypes are not yet known to elicit biological differences in the plant or insect hosts. These apparently stable haplotypes suggest separate bacterial populations of long standing.     

Spatial heterogeneity,incidence-incidence and incidence-lesion density relationship of apple scab (<Emphasis Type="Italic">Venturia inaequalis</Emphasis>) in managed orchards

The spatial pattern of apple scab was characterized using 10 years of disease incidence and lesion density data collected in managed orchards located in Quebec, Canada. Distributional analyses indicated that scab incidence was better characterized by the beta-binomial than the binomial distribution in 53 and 65% of the data sets at the leaf and shoot scales, respectively. Median values of the beta-binomial parameter θ, a measure of small-scale aggregation, were near 0 (0.003 and 0.028) at both sampling scales, indicating that disease incidence was close to being randomly distributed (low degree of aggregation). For lesion density, the negative binomial distribution fitted the data better than the Poisson distribution in 86% of the data sets at the leaf scale. The median value of the index of dispersion k was 0.068, indicating that aggregation was present. For all apple scab measurements, the power law models provided a good fit to the data. The estimated slope and intercept parameters were significantly greater than 1 and 0, respectively, suggesting that spatial heterogeneity changed systematically with disease incidence. Results of a covariance analysis showed that spatial heterogeneity of scab incidence at both scales and lesion density was not dependent upon shoot type but that spatial heterogeneity of scab incidence and lesion density at the leaf scale was influenced by the sampling period. A hierarchical analysis showed that scab incidence at the tree scale increased as a saturation-type curve with respect to incidence at the leaf or shoot scales. A similar relationship was observed for incidences at the shoot and leaf scales. An effective sample size model based on the binary power law parameters (Madden and Hughes, Phytopathology 89:770–781, 1999) gave the best fit to the leaf and shoot data, respectively. The incidence-lesion density relationship at both scales was well described by a complementary log-log (CLL) and log transformation model ( R_adj² = 0.97 and R_adj² = 0.94 ) \left( {R_{{adj}}^2 = 0.97\,and\,R_{{adj}}^2 = 0.94} \right) , however, the models tended to underestimate lesion density. The information of the spatial relations of apple scab within and between hierarchical scales acquired from this study can be used in developing and evaluating practical disease management strategies and to improve apple scab assessments for fungicide or cultivar susceptibility trials.     

PCR-based detection of sunflower white blister rust (<Emphasis Type="Italic">Pustula helianthicola</Emphasis> C. Rost &; Thines) in soil samples and asymptomatic host tissue

Sequencing of partial cox2 (part of the mitochondrial cytochrome-c-oxydase (COX) gene) was performed with samples from the oomycete genus Pustula, the white blister rusts of Asteraceae and related families. Sequence comparison uncovered several single nucleotide polymorphisms (SNPs) between P. spinulosa and host specific strains of Pustula isolated from Senecio vulgaris, Tragopogon pratensis and cultivated sunflower, Helianthus annuus. Based on these differences, specific primers were designed for PCR-based detection of white blister rust strains pathogenic to sunflower. The specificity of the primers was confirmed by cross testing with DNA from various oomycetes occurring in the same locality. The limit of detection for DNA of P. helianthicola was 10 pg. This allowed detection with DNA from single sporangia and single oospores. The PCR-based experiments allowed detection of the presence of sunflower white blister rust in soil samples from fields on which infected plants had been cultivated several months before. Moreover, the molecular tools were successfully applied to trace the pathogen in asymptomatic tissue of infected plants, demonstrating the systemic nature of Pustula on sunflower.     

Pathogenic Botryosphaeriaceae associated with <Emphasis Type="Italic">Mangifera indica</Emphasis> in the Kimberley Region of Western Australia

Members of the Botryosphaeriaceae, in particular Lasiodiplodia theobromae, Neofusicoccum parvum, N. mangiferum and Botryosphaeria dothidea, commonly cause stem cankers, dieback and stem end rot of mangoes worldwide. In the current study, eight taxa of Botryosphaeriaceae were identified as canker-associated fungi, pathogens, potential pathogens or endophytes of mangoes in the Kimberley, Australia. These include Neoscytalidium novaehollandiae, Ne. dimidiatum, Pseudofusicoccum adansoniae, P. ardesiacum, P. kimberleyense, Lasiodiplodia sp. 1, L. iraniensis and L. pseudotheobromae. The pathogenicity of a selection of these species toward fruit and branches was tested. All were pathogenic to mango in comparison to the control, with Lasiodiplodia spp. being the most pathogenic. It appears that either geographic isolation or the unique growing conditions in the Kimberley may have provided an effective barrier to the acquisition or establishment of known botryosphaeriaceous pathogens. Wounds caused by mechanical pruning may provide an entry point for infection, whilst severe pruning may increase plant stress.     

ROS and NO production in compatible and incompatible tomato-<Emphasis Type="Italic">Meloidogyne incognita</Emphasis> interactions

Nitric oxide (NO) has been shown to be an essential regulatory molecule in plant response to pathogen infection in synergy with reactive oxygen species (ROS). At the present, nothing is known about the role of NO in disease resistance to nematode infection. We used a resistant tomato cultivar with different sensitivity to avirulent and virulent populations of the root-knot nematode Meloidogyne incognita to investigate the key components involved in oxidative and nitrosative metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, and nitric oxide synthase (NOS)-like and nitrate reductase activities, as potential sources of NO. A rapid NO accumulation and ROS production were found at 12 h after infection in compatible and incompatible tomato-nematode interactions, whereas the amount of NO and ROS gave different results 24 and 48 h after infection amongst compatible and incompatible interactions. NOS-like arginine-dependent enzyme rather than nitrate reductase was the main source of NO production, and NOS-like activity increased substantially in the incompatible interaction. We can envisage a functional overlap of both NO and ROS in tomato defence response to nematode invasion, NO and H₂O₂ cooperating in triggering hypersensitive cell death. Therefore, NO and ROS are key molecules which may help to orchestrate events following nematode challenge, and which may influence the host cellular metabolism.     

Pathogenicity of indole-3-acetic acid producing fungus <Emphasis Type="Italic">Fusarium delphinoides</Emphasis> strain GPK towards chickpea and pigeon pea

An indole-3-acetic acid (IAA) producing fungal strain was isolated from chickpea grown rhizospheric soil samples. Based on morphological and Internal Transcribed Spacer (ITS) region sequence analysis the new isolate was identified as Fusarium delphinoides. The Fusarium delphinoides strain produces and secretes IAA in-vitro as identified by HPLC and Mass spectrometry. The IAA production is dependent on tryptophan (Trp) as a nitrogen source in the medium. The IAA production is influenced by growth conditions such as pH of the medium, concentration of Trp and the nature of the carbon source. Additional nitrogen sources repress Trp dependent IAA production. Glucose and Trp served as the best carbon and nitrogen sources respectively. Pathogenicity of Fusarium delphinoides towards the plants was tested by electrolyte, nutrient leakage analysis and also by scoring the disease symptoms. Two cultivars of chickpea (ICCV-10 and L-550) and two cultivars of pigeon pea (Maruti and PT-221) were assessed for the pathogenicity by inoculating with spores of Fusarium delphinoides. The inoculation induced symptoms of Fusarium wilt as in the case of Fusarium oxysporum f. sp. ciceris (FOC), a known pathogen causing Fusarium wilt in chickpea. Electrolyte and nutrient leakage from the infected plants were used to assess the resistance, tolerance (moderately resistance) and susceptibility of the plants to the infection. Based on the results, both the pigeon pea cultivars (Maruti and PT-221) were rated as resistant, and ICCV-10 was rated as a tolerant cultivar of chickpea. However, chickpea cultivar L −550 was found to be a susceptible host for infection by Fusarium delphinoides. These results suggest that Fusarium delphinoides, which belongs to the Fusarium dimerum species group, is an IAA producing plant pathogen and causes wilt in chickpea. Further, along with pathogenicity tests, electrolyte and nutrient leakage analysis can be used to assess the pathogenicity of pathogenic fungi.     

Advances in sunflower downy mildew research

This review summarises the progress in research on sunflower downy mildew as reported in publications of the past 10 to 15 years, the period since the last comprehensive review on Plasmopara halstedii. Particular attention is paid to subjects that showed much progress and may be of particular interest to sunflower pathologists, mycologists or molecular biologists. Accordingly, single sections are devoted to the problems of taxonomic and phylogenetic aspects, host specificity, the host—pathogen interaction including resistance phenomena, as well as epidemiology and disease management. Reflecting the progress achieved in some fields and illuminating the deficits in others should stimulate the reader's interest in this very significant pathosystem.     

Diversity and fitness of <Emphasis Type="Italic">Plasmopara viticola</Emphasis> isolates resistant to QoI fungicides

The effectiveness of Quinone outside Inhibitor (QoI) fungicides against grape downy mildew in European vineyards has significantly decreased in the last decade. One nucleotide polymorphism, G143A in the cytochrome b gene of Plasmopara viticola, is involved in resistance to QoIs. Previous genetic examination on the mitochondrial genomes showed four major haplotypes (IR, IS, IIR, IIS) coexisting in European vineyards. A resistant allele (G143A) was present in IR and IIR haplotypes. The purpose of the present study was to estimate the diversity of the different mitochondrial haplotypes and their distribution in QoI-resistant populations before evaluating the potential cost of the resistant mutation G143A in P. viticola population. From 2000 to 2004, the frequencies of resistant isolates ranged from 0% to 23.25% with an average of 4.64 % among the populations examined. To evaluate the fitness of sensitive and resistant isolates, a comparison of different biological parameters including latent period, spore production and infection frequency was performed, enabling a fitness index (F_I) to be determined. Resistant isolates exhibited greater infection frequency than sensitive isolates, whereas no significant difference was found in sporulation ability and latent period between sensitive and resistant isolates. To further investigate competitiveness among isolates, an assay including two resistant isolates in different proportion with a sensitive isolate was conducted on eight asexual growing cycles in the absence of a QoI fungicide. The competitiveness of resistant isolates varied according to their fitness parameters, suggesting that there is no noticeable cost of QoI resistance in controlled conditions in Plasmopara viticola.