Real‐time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds

Fusarium oxysporum f. sp. phaseoli (Fop) is a devastating pathogen that can cause significant economic losses and can be introduced into fields through infested Phaseolus vulgaris (common bean) seeds. Efficient seed health testing methods can aid in preventing long‐distance dissemination of this pathogen by contaminated seeds. In order to improve detection of Fop in seed, a rapid, accurate and sensitive real‐time PCR assay (qPCR) protocol was developed for detection of Fop in common bean seeds. Seed lots of seven cultivars with infection incidence ranging from 0·25 to 20% were prepared by mixing known amounts of Fop‐infected seeds with Fop‐free seeds. Direct comparisons between SYBR Green and TaqMan qPCR methods were performed using primers based on the Fop virulence factor ftf1. The primers developed in this study produced a 63 bp product for highly virulent strains of Fop but did not produce an amplicon for nonpathogenic or weakly pathogenic isolates of F. oxysporum from P. vulgaris or other hosts. Under optimized conditions, both qPCR assays detected Fop infection at low levels (0·25%); however, the results suggest the TaqMan assay was more reliable at quantification than the SYBR Green assay. Linear regression models were fitted to the relationships between results of qPCR assays and infection incidence, but the models differed among cultivars. Fungal biomass per seed differed among cultivars and was related to seed size. The results indicate that the TaqMan assay developed in this study is a useful tool for the detection and quantification of Fop in bean seeds.     

Quantification of Mirafiori lettuce big‐vein virus and its vector,Olpidium virulentus,from soil using real‐time PCR

Big vein disease of lettuce (Lactuca sativa) is an economically important disease transmitted through soil by Olpidium virulentus, and has occurred in most production areas worldwide. The disease is assumed to be caused by Mirafiori lettuce big‐vein virus (MiLBVV). To understand the dynamics of the virus and its vector, MiLBVV and O. virulentus were directly detected in soil. DNA and RNA were extracted from 5 g soil using a bead beating method, followed by purification using adsorption to a column. Detection and quantification were performed using real‐time PCR and a TaqMan probe that was prepared based on the CP region of MiLBVV and the rDNA‐ITS region of O. virulentus, respectively. Furthermore, using a visual assessment of the incidence rate of big vein disease on lettuce in agricultural fields, the C_t values of MiLBVV and O. virulentus from soil were also determined using real‐time PCR. The results showed that MiLBVV concentrations in the soil were high in the field, as also determined by a visual assessment of the incidence rate of big vein disease on lettuce. However, the amount of O. virulentus in soil was not directly correlated with the incidence of MiLBVV. From these results, it is suggested that the risk of lettuce crops developing big vein disease can be estimated using an index of the amount of MiLBVV in the soil.     

Simultaneous monitoring and quantification of Melampsora allii‐populina and Melampsora larici‐populina on infected poplar leaves using a duplex real‐time PCR assay

The rust fungi Melampsora larici‐populina (Mlp) and Melampsora allii‐populina (Map) are the main phytosanitary constraints for commercial poplar cultivation in Europe. Although Mlp is more aggressive and prevalent than Map, the two species may co‐infect the same poplar tree or even the same poplar leaf, making the epidemiological surveys of each species difficult to achieve. In this study, a new duplex real‐time PCR assay targeting each species was developed, based on single‐copy genes. This test proved to be specific, inclusive and was successfully used to detect and quantify each species, starting from urediniospore samples or directly from infected poplar leaves, with or without visible symptoms. This new molecular tool was also assessed for comparative studies of time‐course infection experiments on artificially inoculated poplar leaf discs. These studies showed that the growth dynamics of Map were significantly slower when the two species were co‐inoculated on the same leaf disc, confirming that Map is less aggressive than Mlp.     

Detection and quantification of airborne inoculum of Hymenoscyphus pseudoalbidus using real‐time PCR assays

A method based on real‐time polymerase chain reaction (PCR) and the use of rotating‐arm spore traps was developed for quantifying airborne Hymenoscyphus pseudoalbidus ascospores. The method was sensitive and reproducible, and the collection efficiency was 10% of the spores present in the air. The temporal ascospore dispersal pattern was studied over 3 years by collecting spores every 15 days for a 24 h air‐sampling period during the ash‐growing season. The highest production was detected from the end of June to the beginning of September. The overall ascospore production did not differ significantly among stands within a specific year but there were differences from year to year. There was a positive correlation between air temperature and the number of ascospores trapped, with most of the positive samples being observed at temperatures above 12°C. The vertical profile of ascospore dispersal showed a strong decrease in ascospore density within a height of 3 m, regardless of date of collection. An analysis of the spore traps installed at increasing distances from an infected stand showed that most of the ascospores were deposited downwind within 50 m of the stand. These data are discussed in context of the epidemiology of the disease.     

Development of a quantitative real‐time PCR assay for Phytophthora infestans and its applicability to leaf,tuber and soil samples

A sensitive real‐time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non‐potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.     

Real‐time and qualitative PCR for detecting Pseudomonas syringae pv. actinidiae isolates causing recent outbreaks of kiwifruit bacterial canker

Since 2008, Pseudomonas syringae pv. actinidiae virulent strains (Psa‐V) have quickly spread across the main areas of kiwifruit (Actinidia deliciosa and A. chinensis) cultivation causing sudden and re‐emerging outbreaks of bacterial canker to both species. The disease caused by Psa‐V strains is considered worldwide as pandemic. Recently, P. syringae strains (ex Psa‐LV, now called PsD) phylogenetically related to Psa‐V have been isolated from kiwifruit, but cause only minor damage (i.e. leaf spot) to the host. The different biological significance of these bacterial populations affecting kiwifruit highlights the importance of having a diagnostic method able to detect Psa‐V, which is currently solely responsible for the severe damage to the kiwifruit industry. In order to improve the specific molecular detection of Psa‐V, a real‐time PCR assay has been developed based on EvaGreen chemistry, together with a novel qualitative PCR (PCR‐C). Both methods are based on specific primer sets for the hrpW gene of Psa. The real‐time PCR and PCR‐C were highly specific, detecting down to 50 and 200 fg, respectively, and were applied to a range of organs/tissues of kiwifruit with and without symptoms. These methods are important tools for both sanitary and certification programmes, and will help to avoid the spread of Psa‐V and to check possible inoculum sources. In addition to being used as routine tests, they will also enable the study of the biology of Psa‐V and the disease that it causes, whilst avoiding the detection of other populations of related P. syringae present in kiwifruit.     

Bleeding canker of horse chestnut (Aesculus hippocastanum) in Ireland: incidence,severity and characterization using DNA sequences and real‐time PCR

A survey of bleeding canker disease, caused by Pseudomonas syringae pv. aesculi, was undertaken across Ireland. Incidence has become severe and can be considered epidemic, as 61% of the 1587 horse chestnut trees surveyed showed symptoms of the disease. Bacteria were isolated from a sample of trees and characterized using gyrBDNA sequencing. DNA was also extracted directly from wound tissue. The Irish P. syringae pv. aesculi genotype was identical to genotypes previously sequenced with gyrB from the UK and some other locations in Europe. Real‐time PCR, using existing primers and a newly designed, more pathovar‐specific primer set, was assessed for use in disease screening. With molecular screening, a total of 11 trees from a sample of 55 tested positive for P. syringae pv. aesculi in Ireland. It was more efficient to extract DNA directly from wound tissue, especially fresh bark, for disease detection than to undertake bacterial isolation with subsequent molecular analysis. A further set of sequencing primers was developed for the amplification of the gyrB gene from P. syringae pv. aesculi and their specificity was shown using a diverse sample of bacterial isolate DNAs. The study also isolated and identified other bacterial species from diseased material; some of these are known pathogens (Brenneria nigrifluens, P. marginalis and P. syringae) or have previously been identified as potentially beneficial endophytes of host trees (Erwinia billingiae, E. tolentana, P. fluorescens, P. putida and Raoultella).     

Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index,X. diversicaudatum,X. vuittenezi and X. italiae,and for the quantification of X. index numbers

The ectoparasitic dagger nematodes Xiphinema index and Xiphinema diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real‐time PCR, for the specific detection of these species and of the closely related non‐vector species Xiphinema vuittenezi and Xiphinema italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species whatever the nematode population. The specificity of detection and absence of false positive reaction were confirmed in samples of each species mixed with the three other Xiphinema species or mixed with nematodes representative from other genera (non‐plant‐parasitic Dorylaimida, Longidorus sp., Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. index numbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time‐consuming microscopic identification and counting, this real‐time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.     

Efficacy of UV‐C radiation to reduce seedborne anthracnose (Colletotrichum acutatum) from Andean lupin (Lupinus mutabilis)

The potential of UV‐C radiation of Andean lupin (Lupinus mutabilis) seeds to eradicate seedborne infections of anthracnose caused by Colletotrichum acutatum was investigated. UV‐C doses from 0 to 691.2 kJ m^?2 (resulting from 0 to 96 h of exposure time) on disease incidence reduction and germination on artificially and naturally infected seed were evaluated. The degree of incidence reduction and seed germination was dependent on the dose of UV‐C. The UV‐C doses of 86.4 kJ m^?2 and higher reduced incidence from 6% to 7% to undetectable levels, but these UV‐C doses also reduced seed germination. UV‐C can deleteriously affect physiological processes and overall growth. To assess its impact, L. mutabilis seeds irradiated with UV‐C doses of 57.6 and 86.4 kJ m^?2 were grown. Seedlings grown from noninfected seed and UV‐C treated seed showed an increased concentration of chlorophyll and protein contents, as well as an increase in the activation of defence enzymes peroxidase and catalase, in comparison with plants grown from infected seed. UV‐C doses resulted in seed emergence and seedling dry weight rates that were similar to the noninfected control or better than the fungicide control. Moreover, 57.6 kJ m^?2 reduced transmission of the pathogen from seed to the plantlets by 80%, while 86.4 kJ m^?2 apparently eradicated the pathogen, under greenhouse conditions. The use of UV‐C, first reported here, is advantageous for controlling anthracnose in lupin.     

Characterization and epidemiology of Colletotrichum acutatum sensu lato (C. chrysanthemi) causing Carthamus tinctorius anthracnose

In 2012, Colletotrichum isolates were collected from field‐grown safflower (Carthamus tinctorius) crops in central Italy from plants exhibiting typical anthracnose symptoms. Colletotrichum isolates were also collected from seed surfaces and from within seeds. The genetic variability of these isolates was assessed by a multilocus sequencing approach and compared with those from Colletotrichum chrysanthemi and Colletotrichum carthami isolates from different geographic areas and other Colletotrichum acutatum sensu lato‐related isolates. Phylogenetic analysis revealed that all of the strains isolated from C. tinctorius belonged to the species described as C. chrysanthemi, whereas all of the strains belonging to C. carthami had been isolated from Calendula officinalis. Phenotypic characterization of isolates was performed by assessing growth rates at different temperatures, morphology of colonies on potato dextrose agar (PDA) and the size of conidia. All C. chrysanthemi isolates from safflower had similar growth rates at different temperatures, comparable colony morphologies when grown on PDA and conidial sizes consistent with previously described C. chrysanthemi isolates. Pathogenicity tests were performed by artificially inoculating both seeds and plants and confirmed the seedborne nature of this pathogen. When inoculated on plants, C. chrysanthemi caused the typical symptoms of anthracnose on leaves. This is the first record of this pathogen on C. tinctorius in Italy, and it presents an updated characterization of Colletotrichum isolates pathogenic to safflowers in Europe.     

Species of the Colletotrichum gloeosporioides and C. boninense complexes associated with olive anthracnose

The taxonomic status of Colletotrichum gloeosporioides sensu lato (s.l.) associated with olive anthracnose is still undetermined and the pathogenic ability of this species complex is controversial. In the present study, isolates obtained from olive and provisionally identified as C. gloeosporioides s.l. on the basis of morphological and cultural features were reclassified using ITS and TUB2 as DNA barcode markers and referred to seven distinct species, recently separated within C. gloeosporioides (C. aenigma, C. gloeosporioides sensu stricto (s.s.), C. kahawae, C. queenslandicum, C. siamense and C. theobromicola) and C. boninense (C. karstii) species complexes. Furthermore, isolates of C. kahawae were ascribed to the subspecies ciggaro by analysing the GS gene. A single isolate, not in either of these two species complexes, was not identified at the species level. In pathogenicity tests on detached olive drupes some of these species, including C. aenigma, C. kahawae subsp. ciggaro, C. queenslandicum, C. siamense and C. karstii, were shown to be weakly pathogenic. Moreover, they were found very sporadically on olive. In contrast, some isolates of C. gloeosporioides s.s. and isolates of C. theobromicola proved to be virulent on both green and ripening olives. This study gives a better insight into both the aetiology and the epidemiology of olive anthracnose and might have implications for biosecurity and quarantine because C. theobromicola has never been reported in major European olive‐producing countries.     

Hierarchical models for white mould in snap bean

Limitations on the acceptable proportion (incidence) of pods with white mould may lead to the rejection of entire fields of processing snap bean. The low tolerances (no more than 2% to 6% of pods with white mould) are difficult to estimate with sufficient precision in the field when time is limited. These constraints motivated this study of white mould across three spatial hierarchical levels: pods, plants and quadrats consisting of two adjacent plants within rows. Hierarchical relationships are required when designing formal rules for estimating the incidence of pods with white mould from units higher in the spatial hierarchy. Disease assessments were made on all pods from 38 within‐row transects of 40 plants each of the snap bean cultivar Hystyle. Using probability‐based principles, equations were derived and fitted to data on the incidence of white mould on pods (i_pod), plants (i_pad) and quadrats (i_q(2)), which led to a function directly linking i_pod to i_q(2). The variance of i_pod increased with i_q(2), but that may be of little consequence at the lower values of i_q(2) likely to be associated with i_pod values at processor‐set tolerances. For example, at i_q(2) = 0.1 there was a 92% probability that i_pod was less than 0.02. Assessing i_q(2) may be more efficient than directly estimating i_pod because i_q(2) was about an order of magnitude higher than i_pod. Results suggest it may be feasible to design sampling plans for estimating the proportion of pods with white mould from an assessment of the proportion of diseased quadrats.     

Low genetic variability in Sclerotinia sclerotiorum populations from common bean fields in Minas Gerais State,Brazil, at regional,local and micro‐scales

Sclerotinia sclerotiorum, causal agent of white mould, is the most destructive and widely distributed soilborne pathogen of common bean during the autumn–winter season in Brazil. Nevertheless, little is known about the genetic structure of the pathogen population. Microsatellite (SSR) markers and mycelial compatibility groups (MCGs) were used to characterize 118 isolates collected from 20 bean fields located in the most important growing regions of Minas Gerais State (MG). Additionally, the genetic variability among 10 isolates obtained from a single sclerotium was investigated in 10 different sclerotia. Seventy SSR haplotypes and 14 MCGs were identified among the 118 isolates. The genetic differences within bean growing areas accounted for most of the genetic variation (72%). Despite the relatively high genotypic diversity, the SSR loci were at linkage disequilibrium. Moreover, 70% of the isolates were assigned to only two MCGs, and haplotypes of a given MCG were closely related. The discriminant analysis of principal components revealed five groups. There was strong genetic differentiation between isolates collected in one municipality in southern MG when compared to other regions. Common bean resistance to white mould should be assessed with representative isolates of the five genetic groups and, if possible, of the different MCGs detected in the present study. One to five haplotypes were detected among the 10 isolates obtained from a single sclerotium. Therefore, in order to ensure genetic identity of an isolate, hyphal tip or monoascosporic isolates should be used.     

High‐throughput detection of highly benzimidazole‐resistant allele E198A with mismatch primers in allele‐specific real‐time polymerase chain reaction

BACKGROUND: A point mutation often confers resistance of organisms against medical drugs and agricultural pesticides. Allele‐specific nucleotide polymerase chain reaction (ASPCR) and allele‐specific quantitative real‐time PCR using SYBR Green (ASQPCR) are widely and effectively applied to detect and monitor this type of resistance. However, the former is unsuitable for high‐throughput detection, and the latter often reduces the accuracy of detection. RESULTS: In order to decrease background amplification, a rapid and high‐throughput genotyping method with mismatch primers was developed (ASQPCR‐MP) and applied specifically to survey the frequency of the highly benzimidazole‐resistant MBC^HR mutation (E198A) in the β‐tubulin gene of Sclerotinia sclerotiorum (Lib.) de Bary populations. Genomic DNA from 223 sclerotia was analysed. Similar genotype results were also obtained using ASPCR with mismatch primers and a mycelial growth inhibition assay. It was found that ASQPCR‐MP clearly differentiated MBC^HR and benzimidazole‐sensitive MBC^S phenotypes. Moreover, ASQPCR‐MP took less than 6 h to complete. CONCLUSION: ASQPCR‐MP appears suitable for large epidemiological studies involving resistant genotypes and requiring high‐throughout formats. Copyright © 2009 Society of Chemical Industry     

The use of digital image analysis and real‐time PCR fine‐tunes bioassays for quantification of Cercospora leaf spot disease in sugar beet breeding

Cercospora leaf spot, caused by the fungus Cercospora beticola, is a major fungal sugar beet disease worldwide and the cause of significant yield losses. The disease is most successfully countered by the introduction of genetic tolerance into elite sugar beet hybrids. To this end, breeding programmes require high quality biological assays allowing discrimination of minor differences between plants within a segregating population. This study describes the successful implementation of image analysis software in the bioassays for quantification of necrotic lesions at different stages of C. beticola infection, allowing selection on minor phenotypic differences during the sugar beet breeding process for C. beticola resistance. In addition, a real‐time PCR assay was developed for the quantification of C. beticola pathogen biomass in infected beet canopy. The use of both techniques, even in an early stage of infection, fine‐tunes current bioassays, allowing more accurate and efficient selection of resistant breeding material.     

Morphological and molecular diversity of Colletotrichum spp. causing pepper spot and anthracnose of lychee (Litchi chinensis) in Australia

Since the 1980s a new disease has been affecting Australian lychee. Pepper spot appears as small, black superficial lesions on fruit, leaves, petioles and pedicels and is caused by Colletotrichum gloeosporioides, the same fungus that causes postharvest anthracnose of lychee fruit. The aim of this study was to determine if a new genotype of C. gloeosporioides is responsible for the pepper spot symptom. Morphological assessments, arbitrarily‐primed PCR (ap‐PCR) and DNA sequencing studies did not differentiate isolates of C. gloeosporioides from anthracnose and pepper spot lesions. The ap‐PCR identified 21 different genotypes of C. gloeosporioides, three of which were predominant. A specific genotype identified using ap‐PCR was associated with the production of the teleomorph in culture. Analysis of sequence data of ITS and β‐tubulin regions of representative isolates did not group the lychee isolates into a monophyletic clade; however, given the majority of the isolates were from one of three genotypes found using ap‐PCR, the possibility of a lychee specific group of C. gloeosporioides is discussed.     

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.     

A new anthracnose disease of pyrethrum caused by Colletotrichum tanaceti sp. nov

A new pathogen of pyrethrum (Tanacetum cinerariifolium) causing anthracnose was described as Colletotrichum tanaceti based on morphological characteristics and a four‐gene phylogeny consisting of rDNA‐ITS, β‐tubulin (TUB2), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and actin (ACT) gene sequences. The fungus produced perithecia in culture, requiring an opposite mating type isolate in a heterothallic manner. The initial infection strategy on pyrethrum leaves involved the formation of appressoria followed by production of multilobed infection vesicles in the epidermal cells. Infection and colonization then proceeded through thinner secondary hyphae, which resulted in the initial production of water‐soaked lesions followed by black necrotic lesions. The infection process was suggestive of a hemibiotrophic infection strategy. Moreover, phylogenetic analysis clearly showed that C. destructivum, C. higginsianum and C. panacicola were separate species that also had similar intracellular hemibiotrophic infection strategies as C. tanaceti, which all clustered in the C. destructivum complex. Colletotrichum spp. were detected at 1% incidence in seed of 1 of 19 seed lines, indicating the potential for seed as a source of inoculum into crops. Colletotrichum tanaceti was detected in leaf lesions from 11 of 24 pyrethrum fields surveyed between April and July 2012, at a frequency of 1·3–25·0% of lesions. Anthracnose probably contributes to the complex of foliar diseases reducing green leaf area in pyrethrum fields in Australia.